Inhibitors of human phosphatidylinositol 3-kinase delta

ABSTRACT

Methods of inhibiting phosphatidylinositol 3-kinase delta isoform (PI3Kδ) activity, and methods of treating diseases, such as disorders of immunity and inflammation, in which PI3Kδ plays a role in leukocyte function are disclosed. Preferably, the methods employ active agents that selectively inhibit PI3Kδ, while not significantly inhibiting activity of other PI3K isoforms. Compounds are provided that inhibit PI3Kδ activity, including compounds that selectively inhibit PI3Kδ activity. Methods of using PI3Kδ inhibitory compounds to inhibit cancer cell growth or proliferation are also provided. Accordingly, the invention provides methods of using PI3Kδ inhibitory compounds to inhibit PI3Kδ-mediated processes in vitro and in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/110,204, filed on Apr. 20, 2005, which is a continuation of U.S.patent application Ser. No. 10/697,912, filed on Oct. 30, 2003, now U.S.Pat. No. 6,949,535, which is a divisional of U.S. patent applicationSer. No. 10/027,591, filed on Oct. 19, 2001, now U.S. Pat. No.6,667,300, which is a continuation-in-part of U.S. patent applicationSer. No. 09/841,341, filed on Apr. 24, 2001, now U.S. Pat. No.6,518,277, which claims the benefit of priority to U.S. ProvisionalPatent Application No. 60/199,655, filed Apr. 25, 2000 and U.S.Provisional Patent Application No. 60/238,057, filed Oct. 5, 2000, whichare hereby incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to phosphatidylinositol 3-kinase(PI3K) enzymes, and more particularly to selective inhibitors of PI3Kactivity and to methods of using such materials.

BACKGROUND OF THE INVENTION

Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity (seeRameh et al., J. Biol Chem, 274:8347-8350 (1999) for a review). Theenzyme responsible for generating these phosphorylated signalingproducts, phosphatidylinositol 3-kinase (PI 3-kinase; PI3K), wasoriginally identified as an activity associated with viral oncoproteinsand growth factor receptor tyrosine kinases that phosphorylatesphosphatidylinositol (PI) and its phosphorylated derivatives at the3′-hydroxyl of the inositol ring (Panayotou et al., Trends Cell Biol2:358-60 (1992)).

The levels of phosphatidylinositol-3,4,5-triphosphate (PIP3), theprimary product of PI 3-kinase activation, increase upon treatment ofcells with a variety of agonists. PI 3-kinase activation, therefore, isbelieved to be involved in a range of cellular responses including cellgrowth, differentiation, and apoptosis (Parker et al., Current Biology,5:577-99 (1995); Yao et al., Science, 267:2003-05 (1995)). Though thedownstream targets of phosphorylated lipids generated following PI3-kinase activation have not been well characterized, emerging evidencesuggests that pleckstrin-homology domain- and FYVE-fingerdomain-containing proteins are activated when binding to variousphosphatidylinositol lipids (Sternmark et al., J Cell Sci, 112:4175-83(1999); Lemmon et al., Trends Cell Biol, 7:237-42 (1997)). In vitro,some isoforms of protein kinase C (PKC) are directly activated by PIP3,and the PKC-related protein kinase, PKB, has been shown to be activatedby PI 3-kinase (Burgering et al., Nature, 376:599-602 (1995)).

Presently, the PI 3-kinase enzyme family has been divided into threeclasses based on their substrate specificities. Class I PI3Ks canphosphorylate phosphatidylinositol (PI),phosphatidylinositol-4-phosphate, andphosphatidylinositol-4,5-biphosphate (PIP2) to producephosphatidylinositol-3-phosphate (PIP),phosphatidylinositol-3,4-biphosphate, andphosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ksphosphorylate PI and phosphatidylinositol-4-phosphate, whereas Class IIIPI3Ks can only phosphorylate PI.

The initial purification and molecular cloning of PI 3-kinase revealedthat it was a heterodimer consisting of p85 and p110 subunits (Otsu etal., Cell, 65:91-104 (1991); Hiles et al., Cell, 70:419-29 (1992)).Since then, four distinct Class I PI3Ks have been identified, designatedPI3K α, β, δ, and γ, each consisting of a distinct 110 kDa catalyticsubunit and a regulatory subunit. More specifically, three of thecatalytic subunits, i.e., p110α, p110β and p110δ, each interact with thesame regulatory subunit, p85; whereas p110γ interacts with a distinctregulatory subunit, p101. As described below, the patterns of expressionof each of these PI3Ks in human cells and tissues are also distinct.Though a wealth of information has been accumulated in recent past onthe cellular functions of PI 3-kinases in general, the roles played bythe individual isoforms are largely unknown.

Cloning of bovine p110α has been described. This protein was identifiedas related to the Saccharomyces cerevisiae protein: Vps34p, a proteininvolved in vacuolar protein processing. The recombinant p110α productwas also shown to associate with p85α, to yield a PI3K activity intransfected COS-1 cells. See Hiles et al., Cell, 70, 419-29 (1992).

The cloning of a second human p110 isoform, designated p110β, isdescribed in Hu et al., Mol Cell Biol, 13:7677-88 (1993). This isoformis said to associate with p85 in cells, and to be ubiquitouslyexpressed, as p110β mRNA has been found in numerous human and mousetissues as well as in human umbilical vein endothelial cells, Jurkathuman leukemic T cells, 293 human embryonic kidney cells, mouse 3T3fibroblasts, HeLa cells, and NBT2 rat bladder carcinoma cells. Such wideexpression suggests that this isoform is broadly important in signalingpathways.

Identification of the p110δ isoform of PI 3-kinase is described inChantry et al., J Biol Chem, 272:19236-41 (1997). It was observed thatthe human p110δ isoform is expressed in a tissue-restricted fashion. Itis expressed at high levels in lymphocytes and lymphoid tissues,suggesting that the protein might play a role in PI 3-kinase-mediatedsignaling in the immune system. Details concerning the P110δ isoformalso can be found in U.S. Pat. Nos. 5,858,753; 5,822,910; and 5,985,589.See also, Vanhaesebroeck et al., Proc Natl Acad Sci USA, 94:4330-5(1997), and international publication WO 97/46688.

In each of the PI3Kα, β, and δ subtypes, the p85 subunit acts tolocalize PI 3-kinase to the plasma membrane by the interaction of itsSH2 domain with phosphorylated tyrosine residues (present in anappropriate sequence context) in target proteins (Rameh et al., Cell,83:821-30 (1995)). Two isoforms of p85 have been identified, p85α, whichis ubiquitously expressed, and p85β, which is primarily found in thebrain and lymphoid tissues (Volinia et al., Oncogene, 7:789-93 (1992)).Association of the p85 subunit to the PI 3-kinase p110α, β, or δcatalytic subunits appears to be required for the catalytic activity andstability of these enzymes. In addition, the binding of Ras proteinsalso upregulates PI 3-kinase activity.

The cloning of p110γ revealed still further complexity within the PI3Kfamily of enzymes (Stoyanov et al., Science, 269:690-93 (1995)). Thep110γ isoform is closely related to p110α and p110β (45-48% identity inthe catalytic domain), but as noted does not make use of p85 as atargeting subunit. Instead, p110γ contains an additional domain termed a“pleckstrin homology domain” near its amino terminus. This domain allowsinteraction of p110γ with the βγ subunits of heterotrimeric G proteinsand this interaction appears to regulate its activity.

The p101 regulatory subunit for PI3Kgamma was originally cloned inswine, and the human ortholog identified subsequently (Krugmann et al.,J Biol Chem, 274:17152-8 (1999)). Interaction between the N-terminalregion of p101 with the N-terminal region of p110γ appears to becritical for the PI3Kγ activation through Gβγ mentioned above.

A constitutively active PI3K polypeptide is described in internationalpublication WO 96/25488. This publication discloses preparation of achimeric fusion protein in which a 102-residue fragment of p85 known asthe inter-SH2 (iSH2) region is fused through a linker region to theN-terminus of murine p110. The p85 iSH2 domain apparently is able toactivate PI3K activity in a manner comparable to intact p85 (Klippel etal., Mol Cell Biol, 14:2675-85 (1994)).

Thus, PI 3-kinases can be defined by their amino acid identity or bytheir activity. Additional members of this growing gene family includemore distantly related lipid and protein kinases including Vps34 TOR1,and TOR2 of Saccharomyces cerevisiae (and their mammalian homologs suchas FRAP and mTOR), the ataxia telangiectasia gene product (ATR) and thecatalytic subunit of DNA-dependent protein kinase (DNA-PK). Seegenerally, Hunter, Cell, 83:1-4 (1995).

PI 3-kinase also appears to be involved in a number of aspects ofleukocyte activation. A p85-associated PI 3-kinase activity has beenshown to physically associate with the cytoplasmic domain of CD28, whichis an important costimulatory molecule for the activation of T-cells inresponse to antigen (Pages et al., Nature, 369:327-29 (1994); Rudd,Immunity, 4:527-34 (1996)). Activation of T cells through CD28 lowersthe threshold for activation by antigen and increases the magnitude andduration of the proliferative response. These effects are linked toincreases in the transcription of a number of genes includinginterleukin-2 (IL2), an important T cell growth factor (Fraser et al.,Science, 251:313-16 (1991)). Mutation of CD28 such that it can no longerinteract with PI 3-kinase leads to a failure to initiate IL2 production,suggesting a critical role for PI 3-kinase in T cell activation.

Specific inhibitors against individual members of a family of enzymesprovide invaluable tools for deciphering functions of each enzyme. Twocompounds, LY294002 and wortmannin, have been widely used as PI 3-kinaseinhibitors. These compounds, however, are nonspecific PI3K inhibitors,as they do not distinguish among the four members of Class I PI3-kinases. For example, the IC₅₀ values of wortmannin against each ofthe various Class I PI 3-kinases are in the range of 1-10 nM. Similarly,the IC₅₀ values for LY294002 against each of these PI 3-kinases is about1 μM (Fruman et al., Ann Rev Biochem, 67:481-507 (1998)). Hence, theutility of these compounds in studying the roles of individual Class IPI 3-kinases is limited.

Based on studies using wortmannin, there is evidence that PI 3-kinasefunction also is required for some aspects of leukocyte signalingthrough G-protein coupled receptors (Thelen et al., Proc Natl Acad SciUSA, 91:4960-64 (1994)). Moreover, it has been shown that wortmannin andLY294002 block neutrophil migration and superoxide release. However,inasmuch as these compounds do not distinguish among the variousisoforms of PI3K, it remains unclear which particular PI3K isoform orisoforms are involved in these phenomena.

In view of the above considerations, it is clear that existing knowledgeis lacking with respect to structural and functional features of the PI3-kinase enzymes, including subcellular localization, activation states,substrate affinities, and the like. Moreover, the functions that theseenzymes perform in both normal and diseased tissues remains to beelucidated. In particular, the function of PI3Kδ in leukocytes has notpreviously been characterized, and knowledge concerning its function inhuman physiology remains limited. The coexpression in these tissues ofother PI3K isoforms has heretofore confounded efforts to segregate theactivities of each enzyme. Furthermore, separation of the activities ofthe various PI3K isozymes may not be possible without identification ofinhibitors that demonstrate selective inhibition characteristics.Indeed, Applicants are not presently aware that such selective, orbetter, specific, inhibitors of PI3K isozymes have been demonstrated.

Thus, there exists a need in the art for further structuralcharacterization of the PI3Kδ polypeptide. There also exists a need forfunctional characterization of PI3Kδ. Furthermore, our understanding ofPI3Kδ requires further elaboration of the structural interactions ofp110δ, both with its regulatory subunit and with other proteins in thecell. There also remains a need for selective or specific inhibitors ofPI3K isozymes, in order that the functions of each isozyme can be bettercharacterized. In particular, selective or specific inhibitors of PI3Kδare desirable for exploring the role of this isozyme and for developmentof pharmaceuticals to modulate the activity of the isozyme.

One aspect of the present invention is to provide compounds that caninhibit the biological activity of human PI3Kδ. Another aspect of theinvention is to provide compounds that inhibit PI3Kδ selectively whilehaving relatively low inhibitory potency against the other PI3Kisoforms. Another aspect of the invention is to provide methods ofcharacterizing the function of human PI3Kδ. Another aspect of theinvention is to provide methods of selectively modulating human PI3Kδactivity, and thereby promoting medical treatment of diseases mediatedby PI3Kδ dysfunction. Other aspects and advantages of the invention willbe readily apparent to the artisan having ordinary skill in the art.

SUMMARY OF THE INVENTION

It has now been discovered that these and other aspects can be achievedby the present invention, which, in one aspect, is a method fordisrupting leukocyte function, comprising contacting leukocytes with acompound that selectively inhibits phosphatidylinositol 3-kinase delta(PI3Kδ) activity in the leukocytes. According to the method, theleukocytes can comprise cells selected from the group consisting ofneutrophils, B lymphocytes, T lymphocytes, and basophils.

For example, in cases in which the leukocytes comprise neutrophils, themethod can comprise disrupting at least one neutrophil function selectedfrom the group consisting of stimulated superoxide release, stimulatedexocytosis, and chemotactic migration. Preferably, the method does notsubstantially disrupt bacterial phagocytosis or bacterial killing by theneutrophils. In cases wherein the leukocytes comprise B lymphocytes, themethod can comprise disrupting proliferation of the B lymphocytes orantibody production by the B lymphocytes. In cases wherein theleukocytes comprise T lymphocytes, the method can comprise disruptingproliferation of the T lymphocytes. In cases wherein the leukocytescomprise basophils, the method can comprise disrupting histamine releaseby the basophils.

In the methods of the invention wherein a selective PI3Kδ inhibitor isemployed, it is preferred that the compound be at least about 10-foldselective for inhibition of PI3Kδ relative to other Type I PI3K isoformsin a cell-based assay. More preferably, the compound is at least about20-fold selective for inhibition of PI3Kδ relative to other Type I PI3Kisoforms in a cell-based assay. Still more preferably, the compound isat least about 50-fold selective for inhibition of PI3Kδ relative toother Type I PI3K isoforms in a biochemical assay.

Preferred selective compounds useful according to the methods includecompounds having the structure (I):

wherein A is an optionally substituted monocyclic or bicyclic ringsystem containing at least two nitrogen atoms, and at least one ring ofthe system is aromatic;

X is selected from the group consisting of C(R^(b))₂, CH₂CHR^(b), andCH═C(R^(b));

Y is selected from the group consisting of null, S, SO, SO₂, NH, O,C(═O), OC(═O), C(═O)O, and NHC(═O)CH₂S;

R¹ and R², independently, are selected from the group consisting ofhydrogen, C₁₋₆alkyl, aryl, heteroaryl, halo,NHC(═O)C₁₋₃alkyleneN(R^(a))₂, NO₂, OR^(a), CF₃, OCF₃, N(R^(a))₂, CN,OC(═O)R^(a), C(═O)R^(a), C(═O)OR^(a), arylOR^(b), Het,NR^(a)C(═O)C₁₋₃alkyleneC(═O)OR^(a), arylOC₁₋₃alkyleneN(R^(a))₂,arylOC(═O)R^(a), C₁₋₃alkyleneC(═O)OR^(a), OC₁₋₄alkyleneC(═O)OR^(a),C₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a), C(═O)NR^(a)SO₂R^(a),C₁₋₄alkyleneN(R^(a))₂, C₂₋₆alkenyleneN(R^(a))₂,C(═O)NR^(a)C₁₋₄alkyleneOR^(a), C(═O)NR^(a)C₁₋₄alkyleneHet,OC₂₋₄alkyleneN(R^(a))₂, OC₁₋₄alkyleneCH(OR^(b))CH₂N(R^(a))₂,OC₁₋₄alkyleneHet, OC₂₋₄alkyleneOR^(a), OC₂₋₄alkyleneNR^(a)C(═O)OR^(a),NR^(a)C₁₋₄alkyleneN(R^(a))₂, NR^(a)C(═O)R^(a), NR^(a)C(═O)N(R^(a))₂,N(SO₂C₁₋₄alkyl)₂, NR^(a) (SO₂C₁₋₄alkyl), SO₂N(R^(a))₂, OSO₂CF₃,C₁₋₃alkylenearyl, C₁₋₄alkyleneHet, C₁₋₆alkyleneOR^(b),C₁₋₃alkyleneN(R^(a))₂, C(═O)N(R^(a))₂, NHC(═O)C₁-C₃alkylenearyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, arylOC₁₋₃alkyleneN(R^(a))₂,arylOC(═O)R^(b), NHC(═O)C₁₋₃alkyleneC₃₋₈heterocycloalkyl,NHC(═O)C₁₋₃alkyleneHet, OC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(b),C(═O)C₁₋₄alkyleneHet, and NHC(═O)haloC₁₋₆alkyl;

or R¹ and R² are taken together to form a 3- or 4-membered alkylene oralkenylene chain component of a 5- or 6-membered ring, optionallycontaining at least one heteroatom;

R³ is selected from the group consisting of optionally substitutedhydrogen, C₁₋₆alkyl, C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl,C₁₋₄alkylenecycloalkyl, C₂₋₆alkenyl, C₁₋₃alkylenearyl, arylC₁₋₃alkyl,C(═O)R^(a), aryl, heteroaryl, C(═O)OR^(a), C(═O)N(R^(a))₂,C(═S)N(R^(a))₂, SO₂R^(a)SO₂, N(R^(a))₂, S(═O)R^(a), S(═O)N(R^(a))₂,C(═O)NR^(a)C₁₋₄alkyleneOR^(a), C(═O)NR^(a)C₁₋₄alkyleneHet,C(═O)C₁₋₄alkylenearyl, C(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkylenearyloptionally substituted with one or more of halo, SO₂N(R^(a))₂,N(R^(a))₂, C(═O)OR^(a), NR^(a)SO₂CF₃, CN, NO₂, C(═O)R^(a), OR^(a),C₁₋₄alkyleneN(R^(a))₂, and OC₁₋₄alkyleneN(R^(a))₂,C₁₋₄-alkyleneheteroaryl, C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneC(═O)N(R^(a)) C₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)C(═O)R^(a), C₁₋₄alkyleneOC₁₋₄alkyleneOR^(a),C₁₋₄alkyleneN(R^(a))₂, C₁₋₄alkyleneC(═O)—OR^(a), andC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a);

R^(a) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, C₁₋₃alkyleneN(R^(c))₂, aryl,arylC₁₋₃alkyl, C₁₋₃alkylenearyl, heteroaryl, heteroarylC₁₋₃alkyl, andC₁₋₃alkyleneheteroaryl;

or two R^(a) groups are taken together to form a 5- or 6-membered ring,optionally containing at least one heteroatom;

R^(b) is selected from the group consisting of hydrogen, C₁₋₆alkyl,heteroC₁₋₃alkyl, C₁₋₃alkyleneheteroC₁₋₃alkyl, arylheteroC₁₋₃alkyl, aryl,heteroaryl, arylC₁₋₃alkyl, heteroarylC₁₋₃alkyl, C₁₋₃alkylenearyl, andC₁₋₃alkyleneheteroaryl;

R^(c) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, aryl, and heteroaryl;

Het is a 5- or 6-membered heterocyclic ring, saturated or partially orfully unsaturated, containing at least one heteroatom selected from thegroup consisting of oxygen, nitrogen, and sulfur, and optionallysubstituted with C₁₋₄alkyl or C(═O)OR^(a);

and pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof,

wherein the compound has at least about a 10-fold selective inhibitionfor PI3Kδ relative other Type-I PI3K isoforms in a cell-based assay.

In another embodiment, the invention is a method for treating a medicalcondition mediated by neutrophils, comprising administering to an animalin need thereof an effective amount of a compound that selectivelyinhibits phosphatidylinositol 3-kinase delta (PI3Kδ) activity in theneutrophils. Exemplary medical conditions that can be treated accordingto the method include those conditions characterized by an undesirableneutrophil function selected from the group consisting of stimulatedsuperoxide release, stimulated exocytosis, and chemotactic migration.Preferably, according to the method, phagocytic activity or bacterialkilling by the neutrophils is substantially uninhibited.

In another embodiment, the invention is a method for disrupting afunction of osteoclasts comprising contacting osteoclasts with acompound that selectively inhibits phosphatidylinositol 3-kinase delta(PI3Kγ) activity in the osteoclasts. According to the method, thecompound can comprise a moiety that preferentially binds to bone.

In another embodiment, the invention is a method of ameliorating abone-resorption disorder in an animal in need thereof comprisingadministering to the animal an effective amount of a compound thatinhibits phosphatidylinositol 3-kinase delta (PI3Kγ) activity inosteoclasts of the animal. A preferred bone-resorption disorder amenableto treatment according to the method is osteoporosis.

In another embodiment, the invention is a method for inhibiting thegrowth or proliferation of cancer cells of hematopoietic origin,comprising contacting the cancer cells with a compound that selectivelyinhibits phosphatidylinositol 3-kinase delta (PI3Kγ) activity in thecancer cells. The method can be advantageous in inhibiting the growth orproliferation of cancers selected from the group consisting oflymphomas, multiple myelomas, and leukemias.

In another embodiment, the invention is a method of inhibiting kinaseactivity of a phosphatidylinositol 3-kinase delta (PI3Kγ) polypeptide,comprising contacting the PI3Kγ polypeptide with a compound having thegeneric structure (I).

Preferred compounds useful according to the method include compoundsselected from the group consisting of:

wherein Y is selected from the group consisting of null, S, and NH;

R⁴ is selected from the group consisting of H, halo, NO₂, OH, OCH₃, CH₃,and CF₃;

R⁵ is selected from the group consisting of H, OCH₃, and halo;

or R⁴ and R⁵ together with C-6 and C-7 of the quinazoline ring systemdefine a 5- or 6-membered aromatic ring optionally containing one ormore O, N, or S atoms;

R⁶ is selected from the group consisting of C₁-C₆alkyl, phenyl,halophenyl, alkoxyphenyl, alkylphenyl, biphenyl, benzyl, pyridinyl,4-methylpiperazinyl, C(═O)OC₂H_(s), and morpholinyl;

R^(d), independently, is selected from the group consisting of NH₂,halo, C₁₋₃alkyl, S(C₁₋₃alkyl), OH, NH(C₁₋₃alkyl), N(C₁₋₃alkyl)₂,NH(C₁₋₃alkylenephenyl), and

and

q is 1 or 2,

provided that at least one of R⁴ and R⁵ is other than H when R⁶ isphenyl or 2-chlorophenyl.

More preferably, the compound is selected from the group consisting of:

wherein Y is selected from the group consisting of null, S, and NH;

R⁷ is selected from the group consisting of H, halo, OH, OCH₃, CH₃, andCF₃;

R⁸ is selected from the group consisting of is H, OCH₃, and halo;

or R⁷ and R⁸ together with C-6 and C-7 of the quinazoline ring systemdefine a 5- or 6-membered aromatic ring optionally containing one ormore O, N, or S atoms;

R⁹ is selected from the group consisting of C₁-C₆alkyl, phenyl,halophenyl, alkylphenyl, biphenyl, benzyl, pyridinyl,4-methylpiperazinyl, C(═O)—OC₂H₅, and morpholinyl;

R^(d), independently, is selected from the group consisting of NH₂,halo, C₁₋₃alkyl, S(C₁₋₃alkyl), OH, NH(C₁₋₃alkyl), N(C₁₋₃alkyl)₂,NH(C₁₋₃alkylenephenyl); and

q is 1 or 2,

provided that at least one of R⁷ and R⁸ is different from 6-halo or6,7-dimethoxy groups, and that R⁹ is different from 4-chlorophenyl.

In another embodiment, the invention is a method for disruptingleukocyte function, comprising contacting leukocytes with a compoundhaving a general structure (I).

In another embodiment, the invention is a class of compounds that havebeen observed to inhibit PI3Kδ activity in biochemical and cell-basedassays, and are expected to exhibit therapeutic benefit in medicalconditions in which PI3Kδ activity is excessive or undesirable. Thus,the invention provides a class, of compounds having the structure (II).

Preferably, the compounds have a general structure (IV)

wherein Y is selected from the group consisting of null, S, and NH;

R¹⁰ is selected from the group consisting of H, halo, OH, OCH₃, CH₃, andCF₃;

R¹¹ is selected from the group consisting of H, OCH₃, and halo;

or R¹⁰ and R¹¹ together with C-6 and C-7 of the quinazoline ring systemdefine a 5- or 6-membered aromatic ring optionally containing one ormore O, N, or S atoms;

R¹² is selected from the group consisting of C₁-C₆alkyl, phenyl,halophenyl, alkylphenyl, biphenyl, benzyl, pyridinyl,4-methylpiperazinyl, C(═O)C₂H₅, and morpholinyl;

R^(d), independently, is selected from the group consisting of NH₂,halo, (C₁₋₃alkyl, S(C₁₋₃alkyl), OH, NH(C₁₋₃alkyl), N(C₁₋₃alkyl)₂,NH(C₁₋₃alkylenephenyl), and

q is 1 or 2,

provided that:

(a) at least one of R¹⁰ and R¹¹ is different from 6-halo or6,7-dimethoxy groups;

(b) R¹² is different from 4-chlorophenyl; and

(c) at least one of R¹⁰ and R¹¹ is different from H when R¹² is phenylor 2-chlorophenyl and X is S.

These and other features and advantages of the present invention will beappreciated from the detailed description and examples that are setforth herein. The detailed description and examples are provided toenhance the understanding of the invention, but are not intended tolimit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of a selective PI3Kδ inhibitor of the inventionon the activity of three PI3K isoforms.

FIG. 2 shows the effect of a selective PI3Kδ inhibitor on superoxidegeneration by human neutrophils in the presence of TNF or IgG.

FIG. 3 shows the effect of a selective PI3Kδ inhibitor on superoxidegeneration by human neutrophils in the presence of TNF or fMLP.

FIG. 4 shows the effect of a selective PI3Kδ inhibitor on elastaseexocytosis in the presence of fMLP by human neutrophils.

FIG. 5 shows the effect of a selective PI3Kδ inhibitor on fMLP-inducedchemotaxis by human neutrophils.

FIG. 6 shows that a selective PI3Kδ inhibitor does not affectphagocytosis and killing of S. aureus by neutrophils.

FIG. 7 shows the effect of a selective PI3Kδ inhibitor on proliferationand antibody production by human B lymphocytes.

FIG. 8 shows the effect of a selective PI3Kδ inhibitor on anti-IgMstimulated mouse splenic B lymphocyte proliferation.

FIG. 9 shows the effect of a selective PI3Kδ inhibitor on elastaseexocytosis in an animal model.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides compounds that selectively inhibit the activityof PI3Kδ. The invention further provides methods of inhibiting PI3Kδactivity, including methods of selectively modulating the activity ofthe PI3Kδ isozyme in cells, especially leukocytes, osteoclasts, andcancer cells. The methods include in vitro, in vivo, and ex vivoapplications.

Of particular benefit are methods of selectively modulating PI3Kδactivity in the clinical setting in order to ameliorate disease ordisorders mediated by PI3Kδ activity. Thus, treatment of diseases ordisorders characterized by excessive or inappropriate PI3Kδ activity canbe treated through use of selective modulators of PI3Kδ according to theinvention.

Other methods of the invention include enabling the furthercharacterization of the physiological role of the isozyme. Moreover, theinvention provides pharmaceutical compositions comprising selectivePI3Kδ inhibitors. Also provided are articles of manufacture comprising aselective PI3Kδ inhibitor compound (or a pharmaceutical compositioncomprising the compound) and instructions for using the compound.Details of these and other useful embodiments of the invention are nowdescribed.

The methods described herein benefit from the use of compounds thatselectively inhibit, and preferably specifically inhibit, the activityof PI3Kδ in cells, including cells in vitro, in vivo, or ex vivo. Cellsuseful in the methods include those that express endogenous PI3Kδ,wherein endogenous indicates that the cells express PI3Kδ absentrecombinant introduction into the cells of one or more polynucleotidesencoding a PI3Kδ polypeptide or a biologically active fragment thereof.Methods also encompass use of cells that express exogenous PI3Kδ,wherein one or more polynucleotides encoding PI3Kδ or a biologicallyactive fragment thereof have been introduced into the cell usingrecombinant procedures.

Of particular advantage, the cells can be in vivo, i.e., in a livingsubject, e.g., an animal or human, wherein a PI3Kδ inhibitor can be usedas a therapeutic to inhibit PI3Kδ activity in the subject.Alternatively, the cells can be isolated as discrete cells or in atissue, for ex vivo or in vitro methods. In vitro methods alsoencompassed by the invention can comprise the step of contacting a PI3Kδenzyme or a biologically active fragment thereof with an inhibitorcompound of the invention.

The PI3Kδ enzyme can include a purified and isolated enzyme, wherein theenzyme is isolated from a natural source (e.g., cells or tissues thatnormally express a PI3Kδ polypeptide absent modification by recombinanttechnology) or isolated from cells modified by recombinant techniques toexpress exogenous enzyme.

The term “selective PI3Kδ inhibitor” as used herein refers to a compoundthat inhibits the PI3Kδ isozyme more effectively than other isozymes ofthe PI3K family. A “selective PI3Kδ inhibitor” compound is understood tobe more selective for PI3Kδ than compounds conventionally andgenerically designated PI3K inhibitors, e.g., wortmannin or LY294002.Concomitantly, wortmannin and LY294002 are deemed “nonselective P13Kinhibitors.” Compounds of any type that selectively negatively regulatePI3Kδ expression or activity can be used as selective PI3Kδ inhibitorsin the methods of the invention. Moreover, compounds of any type thatselectively negatively regulate PI3Kδ expression or activity and thatpossess acceptable pharmacological properties can be used as selectivePI3Kδ inhibitors in the therapeutic methods of the invention.

The relative efficacies of compounds as inhibitors of an enzyme activity(or other biological activity) can be established by determining theconcentrations at which each compound inhibits the activity to apredefined extent and then comparing the results. Typically, thepreferred determination is the concentration that inhibits 50% of theactivity in a biochemical assay, i.e., the 50% inhibitory concentrationor determinations can be accomplished using conventional techniquesknown in the art. In general, an IC₅₀ can be determined by measuring theactivity of a given enzyme in the presence of a range of concentrationsof the inhibitor under study. The experimentally obtained values ofenzyme activity then are plotted against the inhibitor concentrationsused. The concentration of the inhibitor that shows 50% enzyme activity(as compared to the activity in the absence of any inhibitor) is takenas the IC₅₀ value. Analogously, other inhibitory concentrations can bedefined through appropriate determinations of activity. For example, insome settings it can be desirable to establish a 90% inhibitoryconcentration, i.e., IC₉₀, etc.

Accordingly, a “selective PI3Kδ inhibitor” alternatively can beunderstood to refer to a compound that exhibits a 50% inhibitoryconcentration (IC₅₀) with respect to PI3Kδ that is at least at least10-fold, preferably at least 20-fold, and more preferably at least30-fold, lower than the IC₅₀ value with respect to any or all of theother Class I PI3K family members. The term “specific PI3Kδ inhibitor”can be understood to refer to a selective PI3Kδ inhibitor compound thatexhibits an IC₅₀ with respect to PI3Kδ that is at least 50-fold,preferably at least 100-fold, more preferably at least 200-fold, andstill more preferably at least 500-fold, lower than the IC₅₀ withrespect to any or all of the other PI3K Class I family members.

Among other things, the invention provides methods of inhibitingleukocyte function. More particularly, the invention provides methods ofinhibiting or suppressing functions of neutrophils and T and Blymphocytes. With respect to neutrophils, it has unexpectedly been foundthat inhibition of PI3Kδ activity inhibits functions of neutrophils. Forexample, it has been observed that the compounds of the invention elicitinhibition of classical neutrophil functions such as stimulatedsuperoxide release, stimulated exocytosis, and chemotactic migration.However, it has been further observed that the method of the inventionpermits suppression of certain functions of neutrophils, while notsubstantially affecting other functions of these cells. For example, ithas been observed that phagocytosis of bacteria by neutrophils is notsubstantially inhibited by the selective PI3Kδ inhibitor compounds ofthe invention.

Thus, the invention includes methods for inhibiting neutrophilfunctions, without substantially inhibiting phagocytosis of bacteria.Neutrophil functions suitable for inhibition according to the methodinclude any function that is mediated by PI3Kδ activity or expression.Such functions include, without limitation, stimulated superoxiderelease, stimulated exocytosis or degranulation, chemotactic migration,adhesion to vascular endothelium (e.g., tethering/rolling ofneutrophils, triggering of neutrophil activity, and/or latching ofneutrophils to endothelium), transmural diapedesis or emigration throughthe endothelium to peripheral tissues. In general, these functions canbe collectively termed “inflammatory functions,” as they are typicallyrelated to neutrophil response to inflammation. The inflammatoryfunctions of neutrophils can be distinguished from the bacterial killingfunctions exhibited by these cells, e.g., phagocytosis and killing ofbacteria. Accordingly, the invention further includes methods oftreating disease states in which one or more of the inflammatoryfunctions of neutrophils are abnormal or undesirable.

It has further been established through the invention that PI3Kδ plays arole in the stimulated proliferation of lymphocytes, including B cellsand T cells. Moreover, PI3Kδ appears to play a role in stimulatedsecretion of antibodies by B cells. Selective PI3Kδ inhibitor compoundsof the invention have been employed to establish that these phenomenacan be abrogated by inhibition of PI3Kδ. Thus, the invention includesmethods of inhibiting lymphocyte proliferation, and methods ofinhibiting antibody production by B lymphocytes. Other methods enabledby the invention include methods of treating disease states in which oneor more of these lymphocyte functions are abnormal or undesirable.

It has now been determined that PI3Kδ activity can be inhibitedselectively or specifically to facilitate treatment of a PI3Kδ-mediateddisease while reducing or eliminating complications that are typicallyassociated with concomitant inhibition of the activity of other Class IPI 3-kinases. To illustrate this embodiment, methods of the inventioncan be practiced using members of a class of compounds that have beenfound to exhibit selective inhibition of PI3Kδ relative to other PI3Kisoforms.

The methods of this embodiment can be practiced using compounds havingthe general structure (III). Preferred methods employ compounds thathave been empirically determined to exhibit at least 10-fold selectiveinhibition of PI3Kδ relative to other PI3K isoforms. For example, themethods can be practiced using the following compounds:

-   3-(2-isopropylphenyl)-5-methyl-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one;-   5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one;-   5-chloro-3-(2-fluorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-fluorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-methoxyphenyl)-5-methyl-2-(9H-purin-y-ylsulfanylmethyl-3H-quinazolin-4-one;-   3-(2,6-dichlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6-fluoro-2-(9h-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   5-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(3-methoxyphenyl-2-(9H-purin-6-ylsulfanylmethyl-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-benzyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-butyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-7-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-morpholin-4-yl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   8-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6,7-difluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-methoxyphenyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   6-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(3-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(9H-purin-6-ylsulfanylmethyl)-3-pyridin-4-yl-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)trifluoromethyl-3H-quinazolin-4-one;-   3-benzyl-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(4-methylpiperazin-1-yl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   3-(2-chlorophenyl)-6-hydroxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   [5-fluoro-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]acetic    acid ethyl ester;-   3-(2,4-dimethoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-biphenyl-2-yl-5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-isopropylphenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-biphenyl-2-yl-5-chloro-3H-quinazolin-4-one;-   5-chloro-3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-fluorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-fluorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-8-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-benzyl-5-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-butyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-morpholin-4-yl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-phenyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-isopropylphenyl)-3H-quinazolin-4-one;    and-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-o-tolyl-3H-quinazolin-4-one.

It has further been determined that the methods of the invention can beadvantageously practiced using members of a class of compounds thatexhibit PI3Kδ inhibitory activity, thereby facilitating inhibitions ofPI3Kδ activity in diseases mediated thereby. For example, in thisembodiment, the methods of the invention can be practiced usingcompounds having the general structure (I).

wherein A is an optionally substituted monocyclic or bicyclic ringsystem containing at least two nitrogen atoms, and at least one ring ofthe system is aromatic;

X is selected from the group consisting of C(R^(b))₂, CH₂CHR^(b), andCH═C(R^(b));

Y is selected from the group consisting of null, S, SO, SO₂, NH, O,C(═O), OC(═O), C(═O)O, and NHC(═O)CH₂S;

R¹ and R², independently, are selected from the group consisting ofhydrogen, C₁₋₃alkyl, aryl, heteroaryl, halo,NHC(═O)C₁₋₃alkyleneN(R^(a))₂, NO₂, OR^(a), CF₃, OCF₃, N(R^(a))₂, CN,OC(═O)R^(a), C(═O)R^(a), C(═O)OR^(a), arylOR^(b), Het,NR^(a)C(═O)C₁₋₃alkyleneC(═O)OR^(a), arylOC₁₋₃alkyleneN(R^(a))₂,arylOC(═O)R^(a), C₁₋₄alkyleneC(═O)OR^(a), OC₁₋₄alkyleneC(═O)OR^(a),C₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a), C(═O)NR^(a)SO₂R^(a),C₁₋₄alkyleneN(R^(a))₂, C₂₋₆alkenyleneN(R^(a))₂,C(═O)NR^(a)C₁₋₄alkyleneOR^(a), C(═O)NR^(a)C₁₋₄alkyleneHet,OC₂₋₄-alkyleneN(R^(a))₂, OC₁₋₄alkyleneCH(OR^(b))CH₂N(R^(a))₂,OC₁₋₄alkyleneHet, OC₂₋₄alkyleneOR^(a), OC₂₋₄alkyleneNR^(a)C(═O)OR^(a),NR^(a)C₁₋₄alkyleneN(R^(a))₂, NR^(a)C(═O)R^(a), NR^(a)C(═O)N(R^(a))N(SO₂C₁₋₄alkyl)₂, NR^(a) (SO₂C₁₋₄alkyl), SO₂N(R^(a))₂, OSO₂CF₃,C₁₋₃alkylenearyl, C₁₋₄alkyleneHet, C₁₋₆alkyleneOR^(b),C₁₋₃alkyleneN(R^(a))₂, C(═O)N(R^(a))₂, NHC(═O)C₁-C₃alkylenearyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, arylOC₁₋₃alkyleneN(R^(a))₂,arylOC(═O)R^(b), NHC(═O)C₁₋₃alkyleneC₃₋₈heterocycloalkyl,NHC(═O)C₁₋₃alkyleneHet, OC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(b),C(═O)C₁₋₄alkyleneHet, and NHC(═O)haloC₁₋₆alkyl;

or R¹ and R² are taken together to form a 3- or 4-membered alkylene oralkenylene chain component of a 5- or 6-membered ring, optionallycontaining at least one heteroatom;

R³ is selected from the group consisting of optionally substitutedhydrogen, C₁₋₄alkyl, C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl,C₁₋₄alkylenecycloalkyl, C₂₋₆alkenyl, C₁₋₃alkylenearyl, arylC₁₋₃alkyl,C(═O)R^(a), aryl, heteroaryl, C(═O) OR^(a), C(═O)N(R^(a))₂,C(═S)N(R^(a)) SO₂R^(a), SO₂N(R^(a))₂, S(═O)R^(a), S(═O)N(R^(a))₂,C(═O)NR^(a)C₁₋₄-alkyleneOR^(a), C(═O)NR^(a)C₁₋₄alkyleneHet,C(═O)C₁₋₄alkylenearyl, C(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkylenearyloptionally substituted with one or more of halo SO₂N(R^(a))₂, N(R^(a))₂,C(═O)OR^(a), NR^(a)SO₂CF₃, CN, NO₂, C(═O)R^(a), OR^(a),C₁₋₄alkyleneN(R^(a))₂, andOC₁₋₄alkyleneN(R^(a))₂/C₁₋₄alkyleneheteroaryl, C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)C₁₋₄alkyleneheteroaryl, (═O)Het,C₁₋₄alkyleneC(═O)—N(R^(a))₂, C₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)C(═O)R^(a), C₁₋₄alkyleneOC₁₋₄alkyleneOR^(a),C₁₋₄alkyleneN(R^(a))₂, C₁₋₄alkyleneC(═O)OR^(a), andC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a);

R^(a) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, C₁₋₃alkyleneN(R^(c))₂, aryl,arylC₁₋₃alkyl, C₁₋₃alkylenearyl, heteroaryl, heteroarylC₁₋₃alkyl, andC₁₋₃alkyleneheteroaryl;

or two R^(a) groups are taken together to form a 5- or 6-membered ring,optionally containing at least one heteroatom;

R^(b) is selected from the group consisting of hydrogen, C₁₋₆alkyl,heteroC₁₋₃alkyl, C₁₋₃alkyleneheteroC₁₋₃alkyl, arylheteroC₁₋₃alkyl, aryl,heteroaryl, arylC₁₋₃alkyl, heteroarylC₁₋₃alkyl, C₁₋₃alkylenearyl, andC₁₋₃alkyleneheteroaryl;

R^(c) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, aryl, and heteroaryl;

Het is a 5- or 6-membered heterocyclic ring, saturated or partially orfully unsaturated, containing at least one heteroatom selected from thegroup consisting of oxygen, nitrogen, and sulfur, and optionallysubstituted with C₁₋₄alkyl or C(═O)OR^(a);

and pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof.

For example, methods of the invention can employ compounds that possessPI3Kδ inhibitory activity, as follows:

-   3-(2-isopropylphenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one;-   5-chloro-3-(2-fluorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-fluorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-methoxyphenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2,6-dichlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3h-quinazolin-4-one;-   5-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-benzyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-butyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-7-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-morpholin-4-yl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   8-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6,7-difluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(3-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   6-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(3-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(9H-purin-6-ylsulfanylmethyl)-3-pyridin-4-yl-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)trifluoromethyl-3H-quinazolin-4-one;-   3-benzyl-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(4-methylpiperazin-1-yl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   3-(2-chlorophenyl)-6-hydroxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   [5-fluoro-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]acetic    acid ethyl ester;-   3-biphenyl-2-yl-5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   5-chloro-3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-isopropylphenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-biphenyl-2-yl-t-chloro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-fluorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-fluorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-8-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-benzyl-5-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-butyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-morpholin-4-yl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-phenyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   3-(4-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6,7-dimethoxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-7-nitro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-6-bromo-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one;-   6-bromo-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-benzo[g]quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-o-tolyl-3H-quinazolin-4-one;    and-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-methoxyphenyl)-3H-quinazolin-4-one.

The invention further provides compounds that are selective inhibitorsof PI3Kδ activity. The compounds exhibit inhibition of PI3Kδ inbiochemical assays, and selectively disrupt function of PI3Kδ-expressingcells in cell-based assays. As described elsewhere herein, the compoundsof the invention have been demonstrated to inhibit certain functions inneutrophils and other leukocytes, as well as functions of osteoclasts.

In general, compounds provided by the invention have the generalstructure (I), a pharmaceutically acceptable salt thereof, or a prodrugthereof:

wherein A is an optionally substituted monocyclic or bicyclic ringsystem containing at least two nitrogen atoms, and at least one ring ofthe system is aromatic;

X is selected from the group consisting of C(R^(b))₂, CH₂CHR^(b), andCH═C(R^(b));

Y is selected from the group consisting of null, S, SO, SO₂, NH, O,C(═O), OC(═O), C(═O)O, and NHC(═O)CH₂S;

R¹ and R², independently, are selected from the group consisting ofhydrogen, C₁₋₆alkyl, aryl, heteroaryl, halo,NHC(═O)C₁₋₃alkyleneN(R^(a))₂, NO₂, OR^(a), CF₃, OCF₃, N(R^(a))₂, CN,OC(═O)R^(a), C(═O)R^(a), C(═O)OR^(a), arylOR^(b), Het,NR^(a)C(═O)C₁₋₃alkyleneC(═O)OR^(a), arylOC₁₋₃-alkyleneN(R^(a))₂,arylOC(═O)R^(a), C₁₋₄alkyleneC(═O)OR^(a), OC₁₋₄alkyleneC(═O)OR^(a),C₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a), C(═O)NR^(a)SO₂R^(a),C₁₋₄alkyleneN(R^(a))₂, C₂₋₆alkenyleneN(R^(a))₂,C(═O)NR^(a)C₁₋₄alkyleneOR^(a), C(═O)NR^(a)C₁₋₄ alkyleneHet,OC₂₋₄alkyleneN(R^(a))₂, OC₂₋₄alkyleneCH(OR^(b))CH₂N(R^(a))₂,OC₁₋₄alkyleneHet, OC₂₋₄alkyleneOR^(a), OC₂₋₄alkyleneNR^(a)C(═O)OR^(a),NR^(a)C₁₋₄alkyleneN(R^(a))₂, NR^(a)C(═O) R^(a), NR^(a)C(═O)N(R^(a))₂,N(SO₂C₁₋₄alkyl)₂, NR^(a)(SO₂C₁₋₄alkyl), SO₂N(R^(a))₂, OSO₂CF₃,C₁₋₃alkylenearyl, C₁₋₄alkyleneHet, C₁₋₆alkyleneOR^(b),C₁₋₃alkyleneN(R^(a)), C(═O)N(R^(a))₂, NHC(═O)C₁-C₃alkylenearyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, arylOC₁₋₃-alkyleneN(R^(a))₂,arylOC(═O)R^(b), NHC(═O)C₁₋₃alkyleneC₃₋₈-heterocycloalkyl,NHC(═O)C₁₋₃alkyleneHet, OC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(b), C(═O)C₁₋₄alkyleneHet, and NHC(═O)haloC₁₋₆ alkyl;

-   -   or R¹ and R² are taken together to form a 3- or 4-membered        alkylene or alkenylene chain component of a 5- or 6-membered        ring, optionally containing at least one heteroatom;

R³ is selected from the group consisting of optionally substitutedhydrogen, C₁₋₆alkyl, C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl,C₁₋₄alkylenecycloalkyl, C₂₋₆alkenyl, C₁₋₃alkylenearyl, arylC₁₋₃alkyl,C(═O)R^(a), aryl, heteroaryl, C(═O)OR^(a), C(═O)N(R^(a))₂,C(═S)N(R^(a))₂, SO₂R^(a), SO₂N(R^(a))₂, S(═O)R^(a), S(═O)N(R^(a))₂,C(═O)NR^(a)C₁₋₄alkyleneOR^(a), C(═O)NR^(a)C₁₋₄alkyleneHet,C(═O)C₁₋₄alkylenearyl, C(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkylenearyloptionally substituted with one or more of halo SO₂N(R^(a))₂, N(R^(a))₂,C(═O)OR^(a), NR^(a)SO₂CF₃, CN, NO₂, C(═O)R^(a), OR^(a),C₁₋₄alkyleneN(R^(a))₂, and OC₁₋₄alkyleneN(R^(a))₂,C₁₋₄-alkyleneheteroaryl, C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)—C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneC(═O)N(R^(a))₂, C₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)C(═O)R^(a), C₁₋₄alkyleneOC₁₋₄alkyleneOR^(a),C₁₋₄alkyleneN(R^(a))₂, C₁₋₄alkyleneC(═O)—OR^(a), andC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a);

R^(a) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, C₁₋₃alkyleneN(R^(c))₂, aryl,arylC₁₋₃alkyl, C₁₋₃alkylenearyl, heteroaryl, heteroarylC₁₋₃alkyl, andC₁₋₃alkyleneheteroaryl;

or two R^(a) groups are taken together to form a 5- or 6-membered ring,optionally containing at least one heteroatom;

R^(b) is selected from the group consisting of hydrogen, C₁₋₆alkyl,heteroC₁₋₃alkyl, C₁₋₃alkyleneheteroC₁₋₃alkyl, arylheteroC₁₋₃alkyl, aryl,heteroaryl, arylC₁₋₃alkyl, heteroarylC₁₋₃alkyl, C₁₋₃alkylenearyl, andC₁₋₃alkyleneheteroaryl;

R^(c) is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₃₋₈cycloalkyl, aryl, and heteroaryl;

Het is a 5- or 6-membered heterocyclic ring, saturated or partially orfully unsaturated, containing at least one heteroatom selected from thegroup consisting of oxygen, nitrogen, and sulfur, and optionallysubstituted with C₁₋₄alkyl or C(═O)OR^(a);

and pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof.

As used herein, the term “alkyl” is defined as straight chained andbranched hydrocarbon groups containing the indicated number of carbonatoms, typically methyl, ethyl, and straight chain and branched propyland butyl groups. The hydrocarbon group can contain up to 16 carbonatoms, preferably one to eight carbon atoms. The term “alkyl” includes“bridged alkyl,” i.e., a C₆-C₁₆ bicyclic or polycyclic hydrocarbongroup, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl,bicyclo-[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl. Theterm “cycloalkyl” is defined as a cyclic C₃-C₈ hydrocarbon group, e.g.,cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.

The term “alkenyl” is defined identically as “alkyl,” except forcontaining a carbon-carbon double bond. “Cycloalkenyl” is definedsimilarly to cycloalkyl, except a carbon-carbon double bond is presentin the ring.

The term “alkylene” is defined as an alkyl group having a substituent.For example, the term “C₁₋₃alkylenearyl” refers to an alkyl groupcontaining one to three carbon atoms, and substituted with an arylgroup.

The term “heteroC₁₋₃alkyl” is defined as a C₁₋₃alkyl group furthercontaining a heteroatom selected from O, S, and NR^(a). For example,—CH₂OCH₃ or —CH₂CH₂SCH₃. The term “arylheteroC₁₋₃alkyl” refers to anaryl group having a heteroC₁₋₃alkyl substituent.

The term “halo” or “halogen” is defined herein to include fluorine,bromine, chlorine, and iodine.

The term “haloalkyl” is defined herein as an alkyl group substitutedwith one or more halo substituents, either fluoro, chloro, bromo, iodo,or combinations thereof. Similarly, “halocycloalkyl” is defined as acycloalkyl group having one or more halo substituents.

The term “aryl,” alone or in combination, is defined herein as amonocyclic or polycyclic aromatic group, preferably a monocyclic orbicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwiseindicated, an “aryl” group can be unsubstituted or substituted, forexample, with one or more, and in particular one to three, halo, alkyl,phenyl, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino,alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl.Exemplary aryl groups include phenyl, naphthyl, biphenyl,tetrahydronaphthyl, chlorophenyl, fluorophenyl, aminophenyl,methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl,carboxyphenyl, and the like. The terms “arylC₁₋₃alkyl” and“heteroaryl(C₁₋₃alkyl” are defined as an aryl or heteroaryl group havinga C₁₋₃alkyl substituent.

The term “heteroaryl” is defined herein as a monocyclic or bicyclic ringsystem containing one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur atom in an aromatic ring, and which can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, substituents, like halo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino,acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Examples ofheteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl,isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl,benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

The term “Het” is defined as monocyclic, bicyclic, and tricyclic groupscontaining one or more heteroatoms selected from the group consisting ofoxygen, nitrogen, and sulfur. A “Het” group also can contain an oxogroup (═O) attached to the ring. Nonlimiting examples of Het groupsinclude 1,3-dioxolane, 2-pyrazoline, pyrazolidine, pyrrolidine,piperazine, a pyrroline, 2H-pyran, 4H-pyran, morpholine, thiopholine,piperidine, 1,4-dithiane, and 1,4-dioxane.

The term “hydroxy” is defined as —OH.

The term “alkoxy” is defined as —OR, wherein R is alkyl.

The term “alkoxyalkyl” is defined as an alkyl group wherein a hydrogenhas been replaced by an alkoxy group. The term “(alkylthio)alkyl” isdefined similarly as alkoxyalkyl, except a sulfur atom, rather than anoxygen atom, is present.

The term “hydroxyalkyl” is defined as a hydroxy group appended to analkyl group.

The term “amino” is defined as —NH₂, and the term “alkylamino” isdefined as —NR₂, wherein at least one R is alkyl and the second R isalkyl or hydrogen.

The term “acylamino” is defined as RC(═O)N, wherein R is alkyl or aryl.

The term “alkylthio” is defined as —SR, wherein R is alkyl.

The term “alkylsulfinyl” is defined as R—SO₂, wherein R is alkyl.

The term “amino” is defined as —NH₂, and the term “alkylamino” isdefined as —NR₂, wherein at least one R is alkyl and the second R isalkyl or hydrogen.

The term “acylamino” is defined as RC(═O)N, wherein R is alkyl or aryl.

The term “alkylthio” is defined as —SR, wherein R is alkyl.

The term “alkylsulfinyl” is defined as R—SO₂, wherein R is alkyl.

The term “alkylsulfonyl” is defined as R—SO₃, wherein R is alkyl.

The term “nitro” is defined as —NO₂.

The term “trifluoromethyl” is defined as —CF₃.

The term “trifluoromethoxy” is defined as —OCF₃.

The term “cyano” is defined as —CN.

In preferred embodiments, X is selected from the group consisting of CH,CH₂CH₂, CH═CH, CH(CHO, CH(CH₂CH₃), CH₂CH(CH₃), and C(CH₃)₂. In furtherpreferred embodiments, Y is selected from the group consisting of null,S, and NH.

The A ring can be monocyclic or bicyclic. Monocyclic A ring systems arearomatic. Bicyclic A ring systems contain at least one aromatic ring,but both rings can be aromatic. Examples of A ring systems include, butare not limited to, imidazolyl, pyrazolyl, 1,2,3-triazolyl, pyridizinyl,pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, purinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,pteridinyl, 1H-indazolyl, and benzimidazolyl.

In a preferred group of compounds of formula (I), A is represented by anoptionally substituted ring system selected from the group consisting of

The A ring system optionally can be substituted with one to three, andpreferably one to two, substituents selected from the group consistingof N(R^(a))₂, halo, C₁₋₃alkyl, S(C₁₋₃alkyl), OR^(a), and

Specific substituents include, but are not limited to, NH₂, NH(CH₃),N(CH₃)₂, NHCH₂C₆H₅, NH(C₂H₅), Cl, F, CH₃, SCH₃, OH, and

In another preferred group of compounds of formula (I), R¹ and R²,independently, are represented by hydrogen, OR^(a), halo, C₁₋₆alkyl,CF₃, NO₂, N(R^(a))₂, NR^(a)C₁₋₃alkyleneN(R^(a))₂, andOC₁₋₃alkyleneOR^(a). Specific substituents include, but are not limitedto, H, OCH₃, Cl, Br, F, CH₃, CF, NO₂, OH, N(CH₃)₂,

and O(CH₂)₂OCH₂C₆H₅. R¹ and R² also can be taken together to form aring, for example, a phenyl ring.

In a preferred embodiment, R³ is selected from the group consisting ofoptionally substituted C₁₋₆alkyl, aryl, heteroaryl, C₃₋₈cycloalkyl,C₃₋₈heterocycloalkyl, C(═O)OR^(a), C₁₋₄alkyleneHet,C₁₋₄alkylenecycloalkyl, C₁₋₄alkylenearyl,C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl, C₁₋₄alkyleneC(═O)OR^(a),C₁₋₄alkyleneC(═O)N(R^(a))₂, C₁₋₄alkyleneC(═O) Het,C₁₋₄alkyleneN(R^(a))₂, and C₁₋₄alkyleneNR^(a)C(═O)R^(a). Specific R³groups include, but are not limited to

The R³ group can be substituted with one to three substituents, forexample, halo, OR^(a), C₁₋₆alkyl, aryl, heteroaryl, NO₂, N(R^(a))₂,NR^(a)SO₂CF₃, NR^(a)C(═O)R^(a), C(═O)OR^(a),N(R^(a))C₁₋₄alkylene(R^(a))₂, SO₂N(R^(a))₂, CN, C(═O)R^(a),C₁₋₄alkyleneN(R^(a))₂, OC₁₋₄alkyleneC≡CR^(a),OC₁₋₄alkyleneC(═O)N(R^(a))₂, OC₁₋₄alkylenearyl, OC₁₋₄alkyleneheteroaryl,OC₁₋₄alkyleneHet, OC₁₋₄alkyleneN(R^(a))₂, andN(R^(a))—C₁₋₄alkyleneN(R^(a))₂. Specific substituents for the R³ groupinclude, but are not limited to, Cl, F, CH₃, CH(CH₃)₂, OH, OCH₃,O(CH₂)₃N(CH₃)₂, OCH₂C≡CH, OCH₂C(═O)—NH₂, C₆H₅, NO₂, NH₂, NHC(═O)CH₃,CO₂H, N(CH₃)CH₂CH₂N—(CH₃)₂, and

As used herein, the quinazoline ring structure, and numbering of thering structure, is

The purine ring structure, and numbering of the ring structure, is

The compounds provided by the invention are exemplified as follows:

-   3-(2-isopropylphenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one;-   5-chloro-3-(2-fluorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-fluorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-methoxyphenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2,6-dichlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3h-quinazolin-4-one;-   5-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-benzyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-butyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-7-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-morpholin-4-yl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   8-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6,7-difluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(3-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   6-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(3-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(9H-purin-6-ylsulfanylmethyl)-3-pyridin-4-yl-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-8-trifluoromethyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-benzyl-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(4-methylpiperazin-1-yl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   3-(2-chlorophenyl)-6-hydroxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   [5-fluoro-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]acetic    acid ethyl ester;-   3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-biphenyl-2-yl-5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   5-chloro-3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-isopropylphenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-biphenyl-2-yl-5-chloro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-fluorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-fluorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-8-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-benzyl-5-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-butyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-morpholin-4-yl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-6-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   3-(4-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6,7-dimethoxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazoline-4-one;-   3-(2-chlorophenyl)-7-nitro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-6-bromo-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one;-   6-bromo-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-benzo[g]quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-o-tolyl-3H-quinazolin-4-one;    and-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-methoxyphenyl)-3H-quinazolin-4-one.

The preferred compounds provided by the invention have the structure(IV), exemplified as follows:

-   3-(2-isopropylphenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one;-   5-chloro-3-(2-fluorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-fluorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2,6-dichlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3h-quinazolin-4-one;-   5-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-benzyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-butyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-7-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-morpholin-4-yl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   8-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-6,7-difluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   6-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(3-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(9H-purin-6-ylsulfanylmethyl)-3-pyridin-4-yl-3H-quinazolin-4-one;-   3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)trifluoromethyl-3H-quinazolin-4-one;-   3-benzyl-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   3-(4-methylpiperazin-1-yl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt;-   3-(2-chlorophenyl)-6-hydroxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   [5-fluoro-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]acetic    acid ethyl ester;-   3-biphenyl-2-yl-5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-isopropylphenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-biphenyl-2-yl-5-chloro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-fluorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-fluorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-8-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-benzyl-5-fluoro-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-butyl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-morpholin-4-yl-3H-quinazolin-4-one;-   2-(6-aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one;    and-   2-(6-aminopurin-9-ylmethyl)-5-chloro-3-o-tolyl-3H-quinazoline-4-one.

It is generally accepted that biological systems can exhibit verysensitive activities with respect to the absolute stereochemical natureof compounds. (See, E. J. Ariens, Medicinal Research Reviews, 6:451-466(1986); E. J. Ariens, Medicinal Research Reviews, 7:367-387 (1987); K.W. Fowler, Handbook of Stereoisomers: Therapeutic Drugs, CRC Press,edited by Donald P. Smith, pp. 35-63 (1989); and S. C. Stinson, Chemicaland Engineering News, 75:38-70 (1997.)

Therefore, the present invention includes all possible stereoisomers andgeometric isomers of compounds of structural formulae (I)-(IV), andincludes not only racemic compounds, but also the optically activeisomers as well. When a compound of structural formulae (I)-(IV) isdesired as a single enantiomer, it can be obtained either by resolutionof the final product or by stereospecific synthesis from eitherisomerically pure starting material or use of a chiral auxiliaryreagent, for example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6),pages 883-888 (1997). Resolution of the final product, an intermediate,or a starting material can be achieved by any suitable method known inthe art. Additionally, in situations where tautomers of the compounds ofstructural formulae (I)-(IV) are possible, the present invention isintended to include all tautomeric forms of the compounds. Specificstereoisomers exhibit an excellent ability to inhibit kinase activity ofPI3Kδ.

The term “prodrug” as used herein refers to compounds that are rapidlytransformed in vivo to a compound having structural formula (I)hereinabove, for example, by hydrolysis. Prodrug design is discussedgenerally in Hardma et al. (Eds.), Goodman and Gilman's ThePharmacological Basis of Therapeutics, 9th ed., pp. 11-16 (1996). Athorough discussion is provided in Higuchi et al., Prodrugs as NovelDelivery Systems, Vol. 14, ASCD Symposium Series, and in Roche (ed.),Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press (1987). Briefly, administration of a drugis followed by elimination from the body or some biotransformationwhereby biological activity of the drug is reduced or eliminated.Alternatively, a biotransformation process can lead to a metabolicby-product, which is itself more active or equally active as compared tothe drug initially administered. Increased understanding of thesebiotransformation processes permits the design of so-called “prodrugs,”which, following a biotransformation, become more physiologically activein their altered state. Prodrugs, therefore, encompass pharmacologicallyinactive compounds that are converted to biologically activemetabolites.

To illustrate, prodrugs can be converted into a pharmacologically activeform through hydrolysis of, for example, an ester or amide linkage,thereby introducing or exposing a functional group on the resultantproduct. The prodrugs can be designed to react with an endogenouscompound to form a water-soluble conjugate that further enhances thepharmacological properties of the compound, for example, increasedcirculatory half-life. Alternatively, prodrugs can be designed toundergo covalent modification on a functional group with, for example,glucuronic acid, sulfate, glutathione, amino acids, or acetate. Theresulting conjugate can be inactivated and excreted in the urine, orrendered more potent than the parent compound. High molecular weightconjugates also can be excreted into the bile, subjected to enzymaticcleavage, and released back into the circulation, thereby effectivelyincreasing the biological half-life of the originally administeredcompound.

Methods for Identifying Negative Regulators of PI3Kδ Activity

The PI3Kδ protein, as well as fragments thereof possessing biologicalactivity, can be used for screening putative negative regulatorcompounds in any of a variety of drug screening techniques. A negativeregulator of PI3Kδ is a compound that diminishes or abolishes theability of PI3Kδ to carry out any of its biological functions. Anexample of such compounds is an agent that decreases the ability of aPI3Kδ polypeptide to phosphorylate phosphatidylinositol or to targetappropriate structures within a cell. The selectivity of a compound thatnegatively regulates PI3Kδ activity can be evaluated by comparing itsactivity on the PI3Kδ to its activity on other proteins. Selectivenegative regulators include, for example, antibodies and other proteinsor peptides that specifically bind to a PI3Kδ polypeptide,oligonucleotides that specifically bind to PI3Kδ polypeptides, and othernonpeptitle compounds (e.g., isolated or synthetic organic molecules)that specifically interact with P13Kδ polypeptides. Negative regulatorsalso include comas described above, but which interact with a specificbinding partner of PI3Kδ polypeptides.

Presently preferred targets for the development of selective negativeregulators of PI3Kδ include, for example:

(1) cytoplasmic regions of PI3Kδ polypeptides that contact otherproteins and/or localize PI3Kδ within a cell;

(2) regions of PI3Kδ polypeptides that bind specific binding partners;

(3) regions of the PI3Kδ polypeptides that bind substrate;

(4) allosteric regulatory sites of the PI3Kδ polypeptides that can orcannot interact directly with the active site upon regulatory signal;

(5) regions of the PI3Kδ polypeptides that mediate multimerization.

For example, one target for development of modulators is the identifiedregulatory interaction of p85 with p110δ, which can be involved inactivation and/or subcellular localization of the p110δ moiety. Stillother selective modulators include those that recognize specificregulatory or PI3Kδ-encoding nucleotide sequences. Modulators of PI3Kδactivity can be therapeutically useful in treatment of a wide range ofdiseases and physiological conditions in which aberrant PI3Kδ activityis involved.

Accordingly, the invention provides methods of characterizing thepotency of a test compound as an inhibitor of PI3Kδ polypeptide, saidmethod comprising the steps of (a) measuring activity of a PI3Kδpolypeptide in the presence of a test compound; (b) comparing theactivity of the PI3Kδ polypeptide in the presence of the test compoundto the activity of the PI3Kδ polypeptide in the presence of anequivalent amount of a reference compound (e.g., a PI3Kδ inhibitorcompound of the invention as described herein), wherein a lower activityof the PI3Kδ polypeptide in the presence of the test compound than inthe presence of the reference indicates that the test compound is a morepotent inhibitor than the reference compound, and a higher activity ofthe PI3Kδ polypeptide in the presence of the test compound than in thepresence of the reference indicates that the test compound is a lesspotent inhibitor than the reference compound.

The invention further provides methods of characterizing the potency ofa test compound as an inhibitor of PI3Kδ polypeptide, comprising thesteps of (a) determining an amount of a control compound (e.g., a PI3Kδinhibitor compound of the invention as described herein) that inhibitsan activity of a PI3Kδ polypeptide by a reference percentage ofinhibition, thereby defining a reference inhibitory amount for thecontrol compound; (b) determining an amount of a test compound thatinhibits an activity of a PI3Kδ polypeptide by a reference percentage ofinhibition, thereby defining a reference inhibitory amount for the testcompound; (c) comparing the reference inhibitory amount for the testcompound to the reference inhibitory amount for the control compound,wherein a lower reference inhibitory amount for the test compound thanfor the control compound indicates that the test compound is a morepotent inhibitor than the control compound, and a higher referenceinhibitory amount for the test compound than for the control compoundindicates that the test compound is a less potent inhibitor than thecontrol compound. In one aspect, the method uses a reference inhibitoryamount which is the amount of the compound than inhibits the activity ofthe PI3Kδ polypeptide by 50%, 60%, 70%, or 80%. In another aspect themethod employs a reference inhibitory amount that is the amount of thecompound that inhibits the activity of the PI3Kδ polypeptide by 90%,95%, or 99%. These methods comprise determining the reference inhibitoryamount of the compounds in an in vitro biochemical assay, in an in vitrocell-based assay, or in an in vivo assay.

The invention further provides methods of identifying a negativeregulator of PI3Kδ activity, comprising the steps of (i) measuringactivity of a PI3Kδ polypeptide in the presence and absence of a testcompound, and (ii) identifying as a negative regulator a test compoundthat decreases PI3Kδ activity and that competes with a compound of theinvention for binding to PI3Kδ. Furthermore, the invention providesmethods for identifying compounds that inhibit PI3Kδ activity,comprising the steps of (i) contacting a PI3Kδ polypeptide with acompound of the invention in the presence and absence of a testcompound, and (ii) identifying a test compound as a negative regulatorof PI3Kδ activity wherein the compound competes with a compound of theinvention for binding to PI3Kδ. The invention therefore provides amethod for screening for candidate negative regulators of PI3Kδ activityand/or to confirm the mode of action of candidate such negativeregulators. Such methods can be employed against other PI3K isoforms inparallel to establish comparative activity of the test compound acrossthe isoforms and/or relative to a compound of the invention.

In these methods, the PI3Kδ polypeptide can be a fragment of p110δ thatexhibits kinase activity, i.e., a fragment comprising the catalytic siteof p110δ. Alternatively, the PI3Kδ polypeptide can be a fragment fromthe p110δ-binding domain of p85 and provides a method to identifyallosteric modulators of PI3Kδ. The methods can be employed in cellsexpressing cells expressing PI3Kδ or its subunits, either endogenouslyor exogenously. Accordingly, the polypeptide employed in such methodscan be free in solution, affixed to a solid support, modified to bedisplayed on a cell surface, or located intracellularly. The modulationof activity or the formation of binding complexes between the PI3Kδpolypeptide and the agent being tested then can be measured.

Human PI3K polypeptides are amenable to biochemical or cell-based highthroughput screening (HTS) assays according to methods known andpracticed in the art, including melanophore assay systems to investigatereceptor-ligand interactions, yeast-based assay systems, and mammaliancell expression systems. For a review, see Jayawickreme and Kost, CurrOpin Biotechnol, 8:629-34 (1997). Automated and miniaturized HTS assaysalso are comprehended as described, for example, in Houston and Banks,Curr Opin Biotechnol, 8:734-40 (1997).

Such HTS assays are used to screen libraries of compounds to identifyparticular compounds that exhibit a desired property. Any library ofcompounds can be used, including chemical libraries, natural productlibraries, and combinatorial libraries comprising random or designedoligopeptides, oligonucleotides, or other organic compounds.

Chemical libraries can contain known compounds, proprietary structuralanalogs of known compounds, or compounds that are identified fromnatural product screening.

Natural product libraries are collections of materials isolated fromnaturals sources, typically, microorganisms, animals, plants, or marineorganisms. Natural products are isolated from their sources byfermentation of microorganisms followed by isolation and extraction ofthe fermentation broths or by direct extraction from the microorganismsor tissues (plants or animal) themselves. Natural product librariesinclude polyketides, nonribosomal peptides, and variants (includingnonnaturally occurring variants) thereof. For a review, see Cane et al.,Science, 282:63-68 (1998).

Combinatorial libraries are composed of large numbers of relatedcompounds, such as peptides, oligonucleotides, or other organiccompounds as a mixture. Such compounds are relatively straightforward todesign and prepare by traditional automated synthesis protocols, PCR,cloning, or proprietary synthetic methods. Of particular interest arepeptide and oligonucleotide combinatorial libraries.

Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries. For a review of combinatorial chemistry andlibraries created thereby, see Myers, Curr Opin Biotechnol, 8:701-07(1997).

Once compounds have been identified that show activity as negativeregulators of PI3Kδ function, a program of optimization can beundertaken in an effort to improve the potency and or selectivity of theactivity. This analysis of structure-activity relationships (SAR)typically involves of iterative series of selective modifications ofcompound structures and their correlation to biochemical or biologicalactivity. Families of related compounds can be designed that all exhibitthe desired activity, with certain members of the family, namely thosepossessing suitable pharmacological profiles, potentially qualifying astherapeutic candidates.

Therapeutic Uses of Inhibitors of PI3Kδ Activity

The invention provides a method for selectively or specificallyinhibiting PI3Kδ activity therapeutically or prophylactically. Themethod comprises administering a selective or specific inhibitor ofPI3Kδ activity in an amount effective therefor. This method can beemployed in treating humans or animals who are or can be subject to anycondition whose symptoms or pathology is mediated by PI3Kδ expression oractivity.

“Treating” as used herein refers to preventing a disorder from occurringin an animal that can be predisposed to the disorder, but has not yetbeen diagnosed as having it; inhibiting the disorder, i.e., arrestingits development; relieving the disorder, i.e., causing its regression;or ameliorating the disorder, i.e., reducing the severity of symptomsassociated with the disorder. “Disorder” is intended to encompassmedical disorders, diseases, conditions, syndromes, and the like,without limitation.

The methods of the invention embrace various modes of treating an animalsubject, preferably a mammal, more preferably a primate, and still morepreferably a human. Among the mammalian animals that can be treated are,for example, companion animals (pets), including dogs and cats; farmanimals, including cattle, horses, sheep, pigs, and goats; laboratoryanimals, including rats, mice, rabbits, guinea pigs, and nonhumanprimates, and zoo specimens. Nonmammalian animals include, for example,birds, fish, reptiles, and amphibians.

In one aspect, the method of the invention can be employed to treatsubjects therapeutically or prophylactically who have or can be subjectto an inflammatory disorder. One aspect of the present invention derivesfrom the involvement of PI3Kδ in mediating aspects of the inflammatoryprocess. Without intending to be bound by any theory, it is theorizedthat, because inflammation involves processes are typically mediated byleukocyte (e.g., neutrophil, lymphocyte, etc.) activation andchemotactic transmigration, and because PI3Kδ can mediate suchphenomena, antagonists of PI3Kδ can be used to suppress injuryassociated with inflammation.

“Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue function. “Inflammatory disorder” also refers to apathological state mediated by influx of leukocytes and/or neutrophilchemotaxis.

“Inflammation” as used herein refers to a localized, protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute, or wall off (sequester) both the injurious agent and the injuredtissue. Inflammation is notably associated with influx of leukocytesand/or neutrophil chemotaxis. Inflammation can result from infectionwith pathogenic organisms and viruses and from noninfectious means suchas trauma or reperfusion following myocardial infarction or stroke,immune response to foreign antigen, and autoimmune responses.Accordingly, inflammatory disorders amenable to the invention encompassdisorders associated with reactions of the specific defense system aswell as with reactions of the nonspecific defense system.

As used herein, the term “specific defense system” refers to thecomponent of the immune system that reacts to the presence of specificantigens. Examples of inflammation resulting from a response of thespecific defense system include the classical response to foreignantigens, autoimmune diseases, and delayed type hypersensitivityresponse mediated by T-cells. Chronic inflammatory diseases, therejection of solid transplanted tissue and organs, e.g., kidney and bonemarrow transplants, and graft versus host disease (GVHD), are furtherexamples of inflammatory reactions of the specific defense system.

The term “nonspecific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, and macrophages).Examples of inflammation that result, at least in part, from a reactionof the nonspecific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents. “Allergic disease” as usedherein refers to any symptoms, tissue damage, or loss of tissue functionresulting from allergy. “Arthritic disease” as used herein refers to anydisease that is characterized by inflammatory lesions of the jointsattributable to a variety of etiologies. “Dermatitis” as used hereinrefers to any of a large family of diseases of the skin that arecharacterized by inflammation of the skin attributable to a variety ofetiologies. “Trans-plant rejection” as used herein refers to any immunereaction directed against grafted tissue, such as organs or cells (e.g.,bone marrow), characterized by a loss of function of the grafted andsurrounding tissues, pain, swelling, leukocytosis, and thrombocytopenia.

The therapeutic methods of the present invention include methods for thetreatment of disorders associated with inflammatory cell activation.

“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclearleukocytes such as neutrophils, basophils, and eosinophils), mast cells,dendritic cells, Langerhans cells, and endothelial cells). It will beappreciated by persons skilled in the art that the activation of one ora combination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory disorder.

The compounds of the invention have been found to inhibit superoxiderelease by neutrophils. Superoxide is released by neutrophils inresponse to any of a variety of stimuli, including signals of infection,as a mechanism of cell killing. For example, superoxide release is knownto be induced by tumor necrosis factor alpha (TNFα), which is releasedby macrophages, mast cells, and lymphocytes upon contact with bacterialcell wall components such as lipopolysaccharide (LPS). TNFα is anextraordinarily potent and promiscuous activator of inflammatoryprocesses, being involved in activation of neutrophils and various othercell types, induction of leukocyte/endothelial cell adhesion, pyrexia,enhanced MHC class I production, and stimulation of angiogenesis.Alternatively, superoxide release can be stimulated byformyl-Met-Leu-Phe (fMLP) or other peptides blocked at the N-terminus byformylated methionine. Such peptides are not normally found ineukaryotes, but are fundamentally characteristic of bacteria, and signalthe presence of bacteria to the immune system. Leukocytes expressing thefMLP receptor, e.g., neutrophils and macrophages, are stimulated tomigrate up gradients of these peptides (i.e., chemotaxis) toward loci ofinfection. As demonstrated herein, the compounds of the inventioninhibit stimulated superoxide release by neutrophils in response toeither TNFα or fMLP. Other functions of neutrophils, includingstimulated exocytosis and directed chemotactic migration, also have beenshown to be inhibited by the PI3Kδ inhibitors of the invention.Accordingly, the compounds of the invention can be expected to be usefulin treating disorders, such as inflammatory disorders, that are mediatedby any or all of these neutrophil functions.

The present invention enables methods of treating such diseases asarthritic diseases, such as rheumatoid arthritis, monoarticulararthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet disease;sepsis, septic shock, endotoxic shock, gram negative sepsis, grampositive sepsis, and toxic shock syndrome; multiple organ injurysyndrome secondary to septicemia, trauma, or hemorrhage; ophthalmicdisorders such as allergic conjunctivitis, vernal conjunctivitis,uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma;pulmonary or respiratory disorders such as asthma, chronic bronchitis,allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g.,chronic obstructive pulmonary disease), silicosis, pulmonarysarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia,bronchiectasis, and pulmonary oxygen toxicity; reperfusion injury of themyocardium, brain, or extremities; fibrosis such as cystic fibrosis;keloid formation or scar tissue formation; atherosclerosis; autoimmunediseases, such as systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud'ssyndrome; and transplant rejection disorders such as GVHD and allograftrejection; chronic glomerulonephritis; inflammatory bowel diseases suchas chronic inflammatory bowel disease (CIBD), Crohn's disease,ulcerative colitis, and necrotizing enterocolitis; inflammatorydermatoses such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjögren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion-associated syndromes; andcytokine-induced toxicity.

The method can have utility in treating subjects who are or can besubject to reperfusion injury, i.e., injury resulting from situations inwhich a tissue or organ experiences a period of ischemia followed byreperfusion. The term “ischemia” refers to localized tissue anemia dueto obstruction of the inflow of arterial blood. Transient ischemiafollowed by reperfusion characteristically results in neutrophilactivation and transmigration through the endothelium of the bloodvessels in the affected area. Accumulation of activated neutrophils inturn results in generation of reactive oxygen metabolites, which damagecomponents of the involved tissue or organ. This phenomenon of“reperfusion injury” is commonly associated with conditions such asvascular stroke (including global and focal ischemia), hemorrhagicshock, myocardial ischemia or infarction, organ transplantation, andcerebral vasospasm. To illustrate, reperfusion injury occurs at thetermination of cardiac bypass procedures or during cardiac arrest whenthe heart, once prevented from receiving blood, begins to reperfuse. Itis expected that inhibition of PI3Kδ activity will result in reducedamounts of reperfusion injury in such situations.

With respect to the nervous system, global ischemia occurs when bloodflow to the entire brain ceases for a period. Global ischemia can resultfrom cardiac arrest. Focal ischemia occurs when a portion of the brainis deprived of its normal blood supply. Focal ischemia can result fromthromboembolytic occlusion of a cerebral vessel, traumatic head injury,edema, or brain tumor. Even if transient, both global and focal ischemiacan cause widespread neuronal damage. Although nerve tissue damageoccurs over hours or even days following the onset of ischemia, somepermanent nerve tissue damage can develop in the initial minutesfollowing the cessation of blood flow to the brain.

Ischemia also can occur in the heart in myocardial infarction and othercardiovascular disorders in which the coronary arteries have beenobstructed as a result of atherosclerosis, thrombus, or spasm.Accordingly, the invention is believed to be useful for treating cardiactissue damage, particularly damage resulting from cardiac ischemia orcaused by reperfusion injury in mammals.

In another aspect, selective inhibitors of PI3Kδ activity, such as thecompounds of the invention, can be employed in methods of treatingdiseases of bone, especially diseases in which osteoclast function isabnormal or undesirable. As shown in Example 6, below, compounds of theinvention inhibit osteoclast function in vitro. Accordingly, the use ofsuch compounds and other PI3Kδ selective inhibitors can be of value intreating osteoporosis, Paget's disease, and related bone resorptiondisorders.

In a further aspect, the invention includes methods of using PI3Kδinhibitory compounds to inhibit the growth or proliferation of cancercells of hematopoietic origin, preferably cancer cells of lymphoidorigin, and more preferably cancer cells related to or derived from Blymphocytes or B lymphocyte progenitors. Cancers amenable to treatmentusing the method of the invention include, without limitation,lymphomas, e.g., malignant neoplasms of lymphoid and reticuloendothelialtissues, such as Burkitt's lymphoma, Hodgkins' lymphoma, non-Hodgkinslymphomas, lymphocytic lymphomas and the like; multiple myelomas; aswell as leukemias such as lymphocytic leukemias, chronic myeloid(myelogenous) leukemias, and the like. In a preferred embodiment, PI3Kδinhibitory compounds can be used to inhibit or control the growth orproliferation of chronic myeloid (myelogenous) leukemia cells.

In another aspect, the invention includes a method for suppressing afunction of basophils and/or mast cells, and thereby enabling treatmentof diseases or disorders characterized by excessive or undesirablebasophil and/or mast cell activity. According to the method, a compoundof the invention can be used that selectively inhibits the expression oractivity of phosphatidylinositol 3-kinase delta (PI3Kδ) in the basophilsand/or mast cells. Preferably, the method employs a PI3Kδ inhibitor inan amount sufficient to inhibit stimulated histamine release by thebasophils and/or mast cells. Accordingly, the use of such compounds andother PI3Kδ selective inhibitors can be of value in treating diseasescharacterized by histamine release, i.e., allergic disorders, includingdisorders such as chronic obstructive pulmonary disease (COPD), asthma,ARDS, emphysema, and related disorders.

Pharmaceutical Compositions of Inhibitors of PI3Kδ Activity

A compound of the present invention can be administered as the neatchemical, but it is typically preferable to administer the compound inthe form of a pharmaceutical composition or formulation. Accordingly,the present invention also provides pharmaceutical compositions thatcomprise a chemical or biological compound (“agent”) that is active as amodulator of PI3Kδ activity and a biocompatible pharmaceutical carrier,adjuvant, or vehicle. The composition can include the agent as the onlyactive moiety or in combination with other agents, such as oligo- orpolynucleotides, oligo- or polypeptides, drugs, or hormones mixed withexcipient(s) or other pharmaceutically acceptable carriers. Carriers andother ingredients can be deemed pharmaceutically acceptable insofar asthey are compatible with other ingredients of the formulation and notdeleterious to the recipient thereof.

Techniques for formulation and administration of pharmaceuticalcompositions can be found in Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing Co, Easton, Pa., 1990. The pharmaceuticalcompositions of the present invention can be manufactured using anyconventional method, e.g., mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping,melt-spinning, spray-drying, or lyophilizing processes. However, theoptimal pharmaceutical formulation will be determined by one of skill inthe art depending on the route of administration and the desired dosage.Such formulations can influence the physical state, stability, rate ofin vivo release, and rate of in vivo clearance of the administeredagent. Depending on the condition being treated, these pharmaceuticalcompositions can be formulated and administered systemically or locally.

The pharmaceutical compositions are formulated to contain suitablepharmaceutically acceptable carriers, and can optionally compriseexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Theadministration modality will generally determine the nature of thecarrier. For example, formulations for parenteral administration cancomprise aqueous solutions of the active compounds in water-solubleform. Carriers suitable for parenteral administration can be selectedfrom among saline, buffered saline, dextrose, water, and otherphysiologically compatible solutions. Preferred carriers for parenteraladministration are physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Fortissue or cellular administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art. For preparations comprisingproteins, the formulation can include stabilizing materials, such aspolyols (e.g., sucrose) and/or surfactants (e.g., nonionic surfactants),and the like.

Alternatively, formulations for parenteral use can comprise dispersionsor suspensions of the active compounds prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, and synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions can contain substances that increase the viscosity of thesuspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.Optionally, the suspension also can contain suitable stabilizers oragents that increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. Aqueous polymers thatprovide pH-sensitive solubilization and/or sustained release of theactive agent also can be used as coatings or matrix structures, e.g.,methacrylic polymers, such as the EUDRAGIT® series available from RohmAmerica Inc. (Piscataway, N.J.). Emulsions, e.g., oil-in-water andwater-in-oil dispersions, also can be used, optionally stabilized by anemulsifying agent or dispersant (surface active materials; surfactants).Suspensions can contain suspending agents such as ethoxylated isostearylalcohols, polyoxy-ethlyene sorbitol and sorbitan esters,micro-crystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, gum tragacanth, and mixtures thereof.

Liposomes containing the active agent also can be employed forparenteral administration. Liposomes generally are derived fromphospholipids or other lipid substances. The compositions in liposomeform also can contain other ingredients, such as stabilizers,preservatives, excipients, and the like. Preferred lipids includephospholipids and phosphatidyl cholines (lecithins), both natural andsynthetic. Methods of forming liposomes are known in the art. See, e.g.,Prescott (Ed.), Methods in Cell Biology, Vol. XIV, p. 33, AcademicPress, New York (1976).

The pharmaceutical compositions comprising the agent in dosages suitablefor oral administration can be formulated using pharmaceuticallyacceptable carriers well known in the art. The preparations formulatedfor oral administration can be in the form of tablets, pills, capsules,cachets, dragees, lozenges, liquids, gels, syrups, slurries, elixirs,suspensions, or powders. To illustrate, pharmaceutical preparations fororal use can be obtained by combining the active compounds with a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Oral formulations can employ liquidcarriers similar in type to those described for parenteral use, e.g.,buffered aqueous solutions, suspensions, and the like.

Preferred oral formulations include tablets, dragees, and gelatincapsules. These preparations can contain one or excipients, whichinclude, without limitation:

a) diluents, such as sugars, including lactose, dextrose, sucrose,mannitol, or sorbitol;

b) binders, such as magnesium aluminum silicate, starch from corn,wheat, rice, potato, etc.;

c) cellulose materials, such as methylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose, polyvinylpyrrolidone,gums, such as gum arabic and gum tragacanth, and proteins, such asgelatin and collagen;

d) disintegrating or solubilizing agents such as cross-linked polyvinylpyrrolidone, starches, agar, alginic acid or a salt thereof, such assodium alginate, or effervescent compositions;

e) lubricants, such as silica, talc, stearic acid or its magnesium orcalcium salt, and polyethylene glycol;

f) flavorants and sweeteners;

g) colorants or pigments, e.g., to identify the product or tocharacterize the quantity (dosage) of active compound; and

h) other ingredients, such as preservatives, stabilizers, swellingagents, emulsifying agents, solution promoters, salts for regulatingosmotic pressure, and buffers.

Gelatin capsules include push-fit capsules made of gelatin, as well assoft, sealed capsules made of gelatin and a coating such as glycerol orsorbitol. Push-fit capsules can contain the active ingredient(s) mixedwith fillers, binders, lubricants, and/or stabilizers, etc. In softcapsules, the active compounds can be dissolved or suspended in suitablefluids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycol with or without stabilizers.

Dragee cores can be provided with suitable coatings such as concentratedsugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.

The pharmaceutical composition can be provided as a salt of the activeagent. Salts tend to be more soluble in aqueous or other protonicsolvents than the corresponding free acid or base forms.Pharmaceutically acceptable salts are well known in the art. Compoundsthat contain acidic moieties can form pharmaceutically acceptable saltswith suitable cations. Suitable pharmaceutically acceptable cationsinclude, for example, alkali metal (e.g., sodium or potassium) andalkaline earth (e.g., calcium or magnesium) cations.

Compounds of structural formula (I) that contain basic moieties can formpharmaceutically acceptable acid addition salts with suitable acids. Forexample, Berge et al. describe pharmaceutically acceptable salts indetail in J Pharm Sci, 66:1 (1977). The salts can be prepared in situduring the final isolation and purification of the compounds of theinvention or separately by reacting a free base function with a suitableacid.

Representative acid addition salts include, but are not limited to,acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorolsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate (isothionate), lactate, maleate,methanesulfonate or sulfate, nicotinate, 2-naphthalenesulfonate,oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate,pivalate, propionate, succinate, tartrate, thiocyanate, phosphate orhydrogen phosphate, glutamate, bicarbonate, p-toluenesulfonate, andundecanoate. Examples of acids that can be employed to formpharmaceutically acceptable acid addition salts include, withoutlimitation, such inorganic acids as hydrochloric acid, hydrobromic acid,sulfuric acid, and phosphoric acid, and such organic acids as oxalicacid, maleic acid, succinic acid, and citric acid.

In light of the foregoing, any reference to compounds of the presentinvention appearing herein is intended to include compounds ofstructural formula (I)-(IV), as well as pharmaceutically acceptablesalts and solvates, and prodrugs, thereof.

Basic addition salts can be prepared in situ during the final isolationand purification of the compounds of the invention or separately byreacting a carboxylic acid-containing moiety with a suitable base suchas the hydroxide, carbonate, or bicarbonate of a pharmaceuticallyacceptable metal cation, or with ammonia or organic primary, secondary,or tertiary amine. Pharmaceutically acceptable basic addition saltsinclude, but are not limited to, cations based on alkali metals oralkaline earth metals such as lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like, and nontoxic quaternaryammonium and amine cations including ammonium, tetramethylammonium,tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium,ethylammonium, diethylammonium, triethylammonium, and the like. Otherrepresentative organic amines useful for the formation of base additionsalts include ethylenediamine, ethanolamine, diethanolamine, piperidine,piperazine, and the like.

Basic nitrogen-containing groups can be quaternized with such agents aslower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl,and diamyl sulfates; long chain alkyl halides such as decyl, lauryl,myristyl, and stearyl chlorides, bromides, and iodides; arylalkylhalides such as benzyl and phenethyl bromides; and others. Productshaving modified solubility or dispersibility are thereby obtained.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier can be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Accordingly, there also is contemplated an article ofmanufacture, such as a container comprising a dosage form of a compoundof the invention and a label containing instructions for use of thecompound. Kits are also contemplated under the invention. For example,the kit can comprise a dosage form of a pharmaceutical composition and apackage insert containing instructions for use of the composition intreatment of a medical condition. In either case, conditions indicatedon the label can include treatment of inflammatory disorders, cancer,etc.

Methods of Administration of Inhibitors of PI3Kδ Activity

Pharmaceutical compositions comprising an inhibitor of PI3Kδ activitycan be administered to the subject by any conventional method, includingparenteral and enteral techniques. Parenteral administration modalitiesinclude those in which the composition is administered by a route otherthan through the gastrointestinal tract, for example, intravenous,intraarterial, intraperitoneal, intramedullary, intramuscular,intraarticular, intrathecal, and intraventricular injections. Enteraladministration modalities include, for example, oral (including buccaland sublingual) and rectal administration. Transepithelialadministration modalities include, for example, transmucosaladministration and transdermal administration. Transmucosaladministration includes, for example, enteral administration as well asnasal, inhalation, and deep lung administration; vaginal administration;and rectal administration. Transdermal administration includes passiveor active transdermal or transcutaneous modalities, including, forexample, patches and iontophoresis devices, as well as topicalapplication of pastes, salves, or ointments. Parenteral administrationalso can be accomplished using a high-pressure technique, e.g.,POWDERJECT®.

Surgical techniques include implantation of depot (reservoir)compositions, osmotic pumps, and the like. A preferred route ofadministration for treatment of inflammation can be local or topicaldelivery for localized disorders such as arthritis, or systemic deliveryfor distributed disorders, e.g., intravenous delivery for reperfusioninjury or for systemic conditions such as septicemia. For otherdiseases, including those involving the respiratory tract, e.g., chronicobstructive pulmonary disease, asthma, and emphysema, administration canbe accomplished by inhalation or deep lung administration of sprays,aerosols, powders, and the like.

For the treatment of neoplastic diseases, especially leukemias and otherdistributed cancers, parenteral administration is typically preferred.Formulations of the compounds to optimize them for biodistributionfollowing parenteral administration would be desirable. The PI3Kδinhibitor compounds can be administered before, during, or afteradministration of chemotherapy, radiotherapy, and/or surgery.

Moreover, the therapeutic index of the PI3Kδ inhibitor compounds can beenhanced by modifying or derivatizing the compounds for targeteddelivery to cancer cells expressing a marker that identifies the cellsas such. For example, the compounds can be linked to an antibody thatrecognizes a marker that is selective or specific for cancer cells, sothat the compounds are brought into the vicinity of the cells to exerttheir effects locally, as previously described (see for example,Pietersz et al., Immunol Rev, 129:57 (1992); Trail et al., Science,261:212 (1993); and Rowlinson-Busza et al., Curr Opin Oncol, 4:1142(1992)). Tumor-directed delivery of these compounds enhances thetherapeutic benefit by, inter alia, minimizing potential nonspecifictoxicities that can result from radiation treatment or chemotherapy. Inanother aspect, PI3Kδ inhibitor compounds and radioisotopes orchemotherapeutic agents can be conjugated to the same anti-tumorantibody.

For the treatment of bone resorption disorders or osteoclast-mediateddisorders, the PI3Kδ inhibitors can be delivered by any suitable method.Focal administration can be desirable, such as by intraarticularinjection. In some cases, it can be desirable to couple the compounds toa moiety that can target the compounds to bone. For example, a PI3Kδinhibitor can be coupled to compounds with high affinity forhydroxyapatite, which is a major constituent of bone. This can beaccomplished, for example, by adapting a tetracycline-coupling methoddeveloped for targeted delivery of estrogen to bone (Orme et al., BioorgMed Chem Lett, 4(11):1375-80 (1994)).

To be effective therapeutically in modulating central nervous systemtargets, the agents used in the methods of the invention should readilypenetrate the blood brain barrier when peripherally administered.Compounds that cannot penetrate the blood brain barrier, however, canstill be effectively administered by an intravenous route.

As noted above, the characteristics of the agent itself and theformulation of the agent can influence the physical state, stability,rate of in vivo release, and rate of in vivo clearance of theadministered agent. Such pharmacokinetic and pharmacodynamic informationcan be collected through preclinical in vitro and in vivo studies, laterconfirmed in humans during the course of clinical trials. Thus, for anycompound used in the method of the invention, a therapeuticallyeffective dose can be estimated initially from biochemical and/orcell-based assays. Then, dosage can be formulated in animal models toachieve a desirable circulating concentration range that modulates PI3Kδexpression or activity. As human studies are conducted, furtherinformation will emerge regarding the appropriate dosage levels andduration of treatment for various diseases and conditions.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe “therapeutic index,” which typically is expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices, i.e., thetoxic dose is substantially higher than the effective dose, arepreferred. The data obtained from such cell culture assays andadditional animal studies can be used in formulating a range of dosagefor human use. The dosage of such compounds lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity.

For the methods of the invention, any effective administration regimenregulating the timing and sequence of doses can be used. Doses of theagent preferably include pharmaceutical dosage units comprising aneffective amount of the agent. As used herein, “effective amount” refersto an amount sufficient to modulate PI3Kδ expression or activity and/orderive a measurable change in a physiological parameter of the subjectthrough administration of one or more of the pharmaceutical dosageunits.

Exemplary dosage levels for a human subject are of the order of fromabout 0.001 milligram of active agent per kilogram body weight (mg/kg)to about 100 mg/kg. Typically, dosage units of the active agent comprisefrom about 0.01 mg to about 10,000 mg, preferably from about 0.1 mg toabout 1,000 mg, depending upon the indication, route of administration,etc. Depending on the route of administration, a suitable dose can becalculated according to body weight, body surface area, or organ size.The final dosage regimen will be determined by the attending physicianin view of good medical practice, considering various factors thatmodify the action of drugs, e.g., the agent's specific activity, theidentity and severity of the disease state, the responsiveness of thepatient, the age, condition, body weight, sex, and diet of the patient,and the severity of any infection. Additional factors that can be takeninto account include time and frequency of administration, drugcombinations, reaction sensitivities, and tolerance/response to therapy.Further refinement of the dosage appropriate for treatment involving anyof the formulations mentioned herein is done routinely by the skilledpractitioner without undue experimentation, especially in light of thedosage information and assays disclosed, as well as the pharmacokineticdata observed in human clinical trials. Appropriate dosages can beascertained through use of established assays for determiningconcentration of the agent in a body fluid or other sample together withdose response data.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agent and the route of administration. Dosage and administration areadjusted to provide sufficient levels of the active moiety or tomaintain the desired effect. Accordingly, the pharmaceuticalcompositions can be administered in a single dose, multiple discretedoses, continuous infusion, sustained release depots, or combinationsthereof, as required to maintain desired minimum level of the agent.Short-acting pharmaceutical compositions (i.e., short half-life) can beadministered once a day or more than once a day (e.g., two, three, orfour times a day). Long acting pharmaceutical compositions might beadministered every 3 to 4 days, every week, or once every two weeks.Pumps, such as subcutaneous, intraperitoneal, or subdural pumps, can bepreferred for continuous infusion.

The following Examples are provided to further aid in understanding theinvention, and presuppose an understanding of conventional methodswell-known to those persons having ordinary skill in the art to whichthe examples pertain, e.g., the construction of vectors and plasmids,the insertion of genes encoding polypeptides into such vectors andplasmids, or the introduction of vectors and plasmids into host cells.Such methods are described in detail in numerous publications including,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989), Ausubel et al. (Eds.),Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994);and Ausubel et al. (Eds.), Short Protocols in Molecular Biology, 4thed., John Wiley & Sons, Inc. (1999). The particular materials andconditions described hereunder are intended to exemplify particularaspects of the invention and should not be construed to limit thereasonable scope thereof.

EXAMPLE 1

Preparation and Purification of Recombinant PI3Kα, β, and δ

Recombinant PI3K heterodimeric complexes 8consisting of a p110 catalyticsubunit and a p85 regulatory subunit were overexpressed using theBAC-TO-BAC.® HT baculovirus expression system (GIBCO/BRL), and thenpurified for use in biochemical assays. The four Class I PI 3-kinaseswere cloned into baculovirus vectors as follows:

p110δ: A FLAG®-tagged version of human p110δ (SEQ ID NO:1) (see Chantryet al., J Biol Chem, 272:19236-41 (1997)) was subcloned using standardrecombinant DNA techniques into the BamH1-Xba1 site of the insect cellexpression vector pFastbac HTb (Life Technologies, Gaithersburg, Md.),such that the clone was in frame with the His tag of the vector. TheFLAG® system is described in U.S. Pat. Nos. 4,703,004; 4,782,137;4,851,341; and 5,011,912, and reagents are available from Eastman KodakCo.

p110α: Similar to the method used for p110δ, described above, aFLAG®-tagged version of p110α (see Volinia et al., Genomics,24(3):427-477 (1994)) was subcloned in BamH1-HindIII sites of pFastbacHTb (Life Technologies) such that the clone was in frame with the Histag of the vector.

p110β: A p110β (see Hu et al., Mol Cell Biol, 13:7677-88 (1993)) clonewas amplified from the human MARATHON® Ready spleen cDNA library(Clontech, Palo Alto Calif.) according to the manufacturer's protocolusing the following primers:

5′ Primer (SEQ ID NO: 3)5′-GATCGAATTCGGCGCCACCATGGACTACAAGGACGACGATGACAAGTGCTTCAGTTTCATAATGCCTCC-3′ 3′ Primer (SEQ ID NO: 4)5′-GATCGCGGCCGCTTAAGATCTGTAGTCTTTCCGAACTGTGTG-3′

The 5′ primer was built to contain a FLAG® tag in frame with the p110βsequence. After amplification, the FLAG®-p110β sequence was subclonedusing standard recombinant techniques into the EcoR1-Not1 sites ofpFastbac HTa (Life Technologies), such that the clone was in frame withthe His tag of the vector.

p110γ: The p110γ cDNA (see Stoyanov et al., Science, 269:690-93 (1995))was amplified from a human Marathon Ready spleen cDNA library (Clontech)according to the manufacturer's protocol using the following primers:

5′ Primer (SEQ ID NO: 5) 5′-AGAATGCGGCCGCATGGAGCTGGAGAACTATAAACAGCCC-3′3′ Primer (SEQ ID NO: 6) 5′-CGCGGATCCTTAGGCTGAATGTTTCTCTCCTTGTTTG-3′A FLAG® tag was subsequently attached to the 5′ end of the p110γsequence and was cloned in the BamH1-Spe1 sites of pFastbac HTb (LifeTechnologies) using standard recombinant DNA techniques, with theFLAG®-110γ sequence in-frame with the His tag of the vector.

p85α: A BamH1-EcoR1 fragment of FLAG®tagged p85 cDNA (see Skolnik etal., Cell, 65:83-89 (1991)) was subcloned into the BamH1-EcoR1 sites ofthe vector pFastbac dual (Life Technologies).

Recombinant baculoviruses containing the above clones were generatedusing manufacturer's recommended protocol (Life Technologies).Baculoviruses expressing His-tagged p110α, p110β, or p110δ catalyticsubunit and p85 subunit were coinfected into Sf21 insect cells. Toenrich the heterodimeric enzyme complex, an excess amount of baculovirusexpressing p85 subunit was infected, and the His-tagged p110 catalyticsubunit complexed with p85 was purified on nickel affinity column. Sincep110γ does not associate with p85, Sf21 cells were infected withrecombinant baculoviruses expressing His-tagged p110γ only. In analternate approach, p101 can be cloned into baculovirus, to permitcoexpression with its preferred binding partner p110γ.

The 72-hour post-infected Sf21 cells (3 liters) were harvested andhomogenized in a hypotonic buffer (20 mM HEPES-KOH, pH 7.8, 5 mM KCl,complete protease inhibitor cocktail (Roche Biochemicals, Indianapolis,Ind.), using a Dounce homogenizer. The homogenates were centrifuged at1,000×g for 15 min. The supernatants were further centrifuged at10,000×g for 20 min, followed by ultracentrifugation at 100,000×g for 60min. The soluble fraction was immediately loaded onto 10 mL of HITRAP®nickel affinity column (Pharmacia, Piscataway, N.J.) equilibrated with50 mL of Buffer A (50 mM HEPES-KOH, pH 7.8, 0.5 M NaCl, 10 mMimidazole). The column was washed extensively with Buffer A, and elutedwith a linear gradient of 10-500 mM imidazole. Free p85 subunit wasremoved from the column during the washing step and only theheterodimeric enzyme complex eluted at 250 mM imidazole. Aliquots ofnickel fractions were analyzed by 10% SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), stained with SYPRO® Red (Molecular Probes,Inc., Eugene, Oreg.), and quantitated with STORM® PhosphoImager(Molecular Dynamics, Sunnyvale, Calif.). The active fractions werepooled and directly loaded onto a 5 mL Hi-trap heparin columnpreequilibrated with Buffer B containing 50 mM HEPES-KOH, pH 7.5, 50 mMNaCl, 2 mM dithiothreitol (DTT). The column was washed with 50 mL ofBuffer B and eluted with a linear gradient of 0.05-2 M NaCl. A singlepeak containing PI3K enzyme complex eluted at 0.8 M NaCl.SDS-polyacrylamide gel analysis showed that the purified PI3K enzymefractions contained a 1:1 stoichiometric complex of p110 and p85subunits. The protein profile of the enzyme complex during heparinchromatography corresponded to that of lipid kinase activity. The activefractions were pooled and frozen under liquid nitrogen.

EXAMPLE 2

PI3Kδ High Throughput Screen (HTS) and Selectivity Assay

A high throughput screen of a proprietary chemical library was performedto identify candidate inhibitors of PI3Kδ activity. PI3Kδ catalyzes aphosphotransfer from γ-[³²P]ATP to PIP₂/PS liposomes at the D3′ positionof the PIP, lipid inositol ring. This reaction is MgCl₂ dependent and isquenched in high molarity potassium phosphate buffer pH 8.0 containing30 mM EDTA. In the screen, this reaction is performed in the presence orabsence of library compounds. The reaction products (and all unlabelledproducts) are transferred to a 96-well, prewetted PVDF filter plate,filtered, and washed in high molarity potassium phosphate. Scintillantis added to the dried wells and the incorporated radioactivity isquantitated.

The majority of assay operations were performed using a BIOMEK® 1000robotics workstations (Beckman) and all plates were read using Wallacliquid scintillation plate counter protocols.

The 3× assay stocks of substrate and enzyme were made and stored in atrough (for robotics assays) or a 96-well, V-bottom, polypropylene plate(for manual assays). Reagents were stable for at least 3 hours at roomtemperature.

The 3× substrate for the HTS contained 0.6 mM Na₂ATP, 0.10 mCi/mLγ-[³²P]ATP (NEN, Pittsburgh, Pa.), 6 μM PIP₂/PS liposomes (Avanti PolarLipids, Inc., Atlanta, Ga.), in 20 mM HEPES, pH 7.4.

The 3× enzyme stock for the HTS contained 1.8 nM P13Kδ, 150 μg/mL horseIgG (used only as a stabilizer), 15 mM MgCl₂, 3 mM DTT in 20 mM HEPES,pH 7.4.

The chemical high throughput screen (HTS) library samples (eachcontaining a pool of 22 compounds) in dimethyl sulfoxide (DMSO) werediluted to 18.75 μM or 37.8 μM in double distilled water, and 20 μL ofthe dilutions were placed in the wells of a 96-well polypropylene platefor assaying. The negative inhibitor control (or positive enzymecontrol) was DMSO diluted in water, and the positive inhibitor controlsemployed concentrations of LY294002 sufficient to provide 50% and 100%inhibition.

To the 20 μL pooled chemical library dilutions, 20 μL of 3× substratewas added. The reaction was initiated with 20 μL of 3× enzyme, incubatedat room temperature for 10 minutes. This dilution established a finalconcentration of 200 μM

ATP in the reaction volume. The reaction was stopped with 150 μL quenchbuffer (1.0 M potassium phosphate pH 8.0, 30 mM EDTA). A portion of thequenched solution (180 μL) was then transferred to a PVDF filter plate(Millipore #MAIP NOB prewetted with sequential 200 μL washes of 100%methanol, water, and finally 1.0 M potassium phosphate pH 8.0 washbuffer).

The PVDF filter plate was aspirated under moderate vacuum (2-5 mm Hg),washed with 5×200 μL of wash buffer, and then dried by aspiration. Thefilter was subsequently blotted, allowed to air dry completely, andinserted into a Wallac counting cassette with 50 μL of Ecoscintscintillation cocktail added per well. The incorporated radioactivitywas quantitated, and data were analyzed, after normalizing to the enzymepositive control (set at 100%), to identify the curve intersection atthe 50% inhibition value to estimate IC₅₀ values for the inhibitors.

A total of 57 pooled master wells were selected for deconvolution, basedon combined criteria of <42% residual activity at the testedconcentration, and a total accepted hit rate of no more than 0.2%. At 22compounds per well, a total of 1254 compounds were identified throughthis deconvolution and individually assayed at the 1× concentration of27.7 μM to identify which compounds exhibited the desired activity. Fromthese assays, 73 compounds were selected and assayed further to developIC₅₀ curves. From the IC₅₀ curve results, 34 compounds were selected forselectivity assays against PI3Kα and PI3Kβ (see assay protocol inExample 11).

From the selectivity assays, one compound,3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(Compound D-000), was selected as being a relatively potent andselective compound. Catalog searches and selectivity assays of manyanalogous compounds of the potent and/or selective hits yielded only onecompound that was both an active and selective analogue of D-000. Thiscompound was purchased from Contract Services Corporation (Catalog#7232154) and differed from D-000 in substituting a phenyl group for the2-chlorophenyl group of D-000.

As described above, the PI 3-kinase inhibitor LY294002 (Calbiochem, LaJolla, Calif.) does not have significant selectivity among the differentPI 3-kinase isoforms tested. Under our assay conditions, LY294002inhibited all three isoforms of PI 3-kinases with an IC₅₀ of 0.3 to 1μM. However, when the compound D-000 was tested against the same PI3-kinase isoforms distinct selectivity was observed.

Specifically, as shown in FIG. 1, D-000 inhibited the activity of the 5isoform of PI3K with an IC₅₀ of approximately 0.3 μM, whereas undersimilar conditions it did not inhibit activities of the α and β isoformsat a limit of 100 μM compound. These results show that D-000 selectivelyinhibits PI3Kδ activity.

EXAMPLES 3-7

Since PI3Kδ is expressed at significant levels only in leukocytes, it isimportant to study the effects of the PI3Kδ-selective inhibitor onleukocyte functions. Accordingly, the effects of PI3Kδ inhibition inseveral types of leukocytes were examined. Neutrophils were examined todetermine the effects that selective inhibition of PI3Kδ might elicit(Example 3, below). It surprisingly was found that selective inhibitionof PI3Kδ activity appears to be significantly associated with inhibitionof some but not all functions characteristic of activated neutrophils.In addition, the effects of PI3Kδ inhibition on B cell and T cellfunction also were tested (Examples 4-5, below). Moreover, as PI3Kδ alsois expressed in osteoclasts, the effect of PI3Kδ inhibition on thefunction of these specialized cells was studied (Example 6, below).

EXAMPLE 3

Characterization of Role of PI3Kδ in Neutrophil Function

The effects of a PI3Kδ inhibitor of the invention, i.e., D-000, onneutrophil functions such as superoxide generation, elastase exocytosis,chemotaxis, and bacterial killing were tested.

A. Preparation of Neutrophils from Human Blood

Aliquots (8 mL) of heparinized blood from healthy volunteers werelayered on 3 mL cushions of 7.3% FICOLL® (Sigma, St. Louis, Mo.) and15.4% HYPAQUE® (Sigma) and centrifuged at 900 rpm for 30 min at roomtemperature in a table top centrifuge (Beckman). The neutrophil-richband just above the FICOLL®-HYPAQUE® cushion was collected and washedwith Hanks' balanced salt solution (HESS) containing 0.1% gelatin.Residual erythrocytes were removed by hypotonic lysis with 0.2% NaCl.The neutrophil preparation was washed twice with HESS containing 0.1%gelatin and used immediately.

B. Measurement of Superoxide Production from Neutrophils

Superoxide generation is one of the hallmarks of neutrophil activation.A variety of activators potentiate superoxide generation by neutrophils.The effect of the PI3Kδ inhibitor D-000 on superoxide generation bythree different agonists: TNFα, IgG, and fMLP, each representingseparate classes of activator, was measured. Superoxide generated by theneutrophils was measured by monitoring the change in absorbance uponreduction of cytochrome C by modification of the method described byGreen et al., (pp. 14.5.1-14.5.11 in Supp. 12, Curr Protocols Immunol(Eds., Colligan et al.) (1994)), as follows. Individual wells of a96-well plate were coated overnight at 4° C. with 50 μL of 2 mg/mLsolution of human fibrinogen or IgG. The wells were washed with PBS andthe following reagents were added to each well: 50 μL of HBSS orsuperoxide dismutase (1 mg/mL), 50 μL of HBSS or TNFα (50 ng/mL), 50 μLcytochrome C (2.7 mg/mL), and 100 μL of purified human neutrophilsuspension (2×10⁶ cells/mL). The plate was centrifuged for 2 min at 200rpm and absorbance at 550 nm was monitored for 2 hr. To measure therelative amounts of superoxide generated, values obtained from thesuperoxide dismutase-containing wells were subtracted from all, andnormalized to the values obtained from the wells without any inhibitor.

As shown in FIG. 2, the PI3Kδ inhibitor D-000 inhibits TNF-inducedsuperoxide generation by neutrophils in a concentration dependentmanner. Superoxide generation induced by TNF was reduced to itshalf-maximal value at about 3 μM D-000. FIG. 2 also reveals thatsuperoxide generation induced by IgG was not significantly inhibited byD-000. In fact, even at 10 μM this PI3Kδ inhibitor did not have anyeffect on superoxide generation induced by IgG.

Next, the effect of D-000 on superoxide generation induced by anotherpotent inducer, the bacterial peptide, formylated-Met-Leu-Phe (fMLP) wasstudied. Like the TNF-induced superoxide generation, fMLP-inducedsuperoxide generation also was inhibited by D-000 (FIG. 3). Theseresults show that the PI3Kδ inhibitor D-000 can prevent stimulusspecific induction of superoxide generation by neutrophils, indicatingthat PI3Kδ is involved in this process.

C. Measurement of Elastase Exocytosis from Neutrophils

In addition to superoxide generation, activated neutrophils also respondby releasing several proteases that are responsible for the destructionof tissues and cartilage during inflammation. As an indication ofprotease release, the effect of D-000 on elastase exocytosis wasmeasured. Elastase exocytosis was quantitated by modification of theprocedure described by Ossanna et al. (J Clin Invest, 77:1939-1951(1986)), as follows. Purified human neutrophils (0.2×10⁶) (treated witheither DMSO or a serial dilution of D-000 in DMSO) were stimulated withfMLP in PBS containing 0.01 mg/mL cytochalasin B, 1.0 μM sodium azide(NaN₃), 5 μg/mL L-methionine and 1 μM fMLP for 90 min at 37° C. in a96-well plate. At the end of the incubation period, the plate wascentrifuged for 5 min at 1000 rpm, and 90 μL of the supernatant wastransferred to 10 μL of 10 mM solution of an elastase substrate peptide,MeO-suc-Ala-Ala-Pro-Val-pNA, wherein MeO-suc=methoxy-succinyl;pNA=p-nitroanilide (Calbiochem, San Diego, Calif.). Absorbance at 410 nmwas monitored for 2 hr in a 96-well plate reader. To measure therelative amounts of elastase excytosed, all absorbance values werenormalized to the values without any inhibitor. As shown in FIG. 4, thePI3Kδ inhibitor D-000 inhibits fMLP-induced elastase exocytosissignificantly, and does so in a dose-dependent fashion. Inhibition washalf-maximal at a concentration of about 2-3 μM D-000.

D. Measurement of fMLP-Induced Human Neutrophil Migration

Neutrophils have the intrinsic capacity to migrate through tissues, andare one of the first cell types to arrive at the sites of inflammationor tissue injury. The effect of D-000 on neutrophil migration towards aconcentration gradient of fMLP was measured. The day before themigration assays were performed, 6-well plates were coated withrecombinant ICAM-1/Fc fusion protein (Van der Vieren et al., Immunity,3:683-690 (1995)) (25 μg/mL in bicarbonate buffer, pH 9.3) and leftovernight at 4° C. After washing, 1% agarose solution, in RPMI-1640 with0.5% bovine serum albumin (BSA), was added to wells with or without aninhibitor, and plates were placed into a refrigerator before punchingholes in the gelled agarose to create plaques (1 central hole surroundedby 6 peripheral ones per well).

Human neutrophils were obtained as described above, and resuspended inRPMI medium supplemented with 0.5% BSA at 5×10⁶ cells/mL. Aftercombining equal volumes of neutrophil suspension and medium (either withDMSO or a serial dilution of the test compound in DMSO), neutrophilswere aliquoted into the peripheral holes, while the central holereceived fMLP (5 μM). Plates were incubated at 37° C. in the presence of5% CO₂ for 4 hr, followed by termination of migration by the addition of1% glutaraldehyde solution in D-PBS. After removing the agarose layer,wells were washed with distilled water and dried.

Analysis of neutrophil migration was conducted on a Nikon DIAPHOT®inverted microscope (1× objective) video workstation using the NIH 1.61program. Using Microsoft Excel and Table Curve 4 (SSPS Inc., ChicagoIll.) programs, a migration index was obtained for each of the studiedconditions. Migration index was defined as the area under a curverepresenting number of migrated neutrophils versus the net distance ofmigration per cell.

As shown in FIG. 5, the PI3Kδ inhibitor D-000 had a profound effect onneutrophil migration, inhibiting this activity in a dose-dependentmanner. The EC₅₀ of this compound for inhibition of neutrophil migrationin this assay is about 1 μM. Based on a visual inspection of therecorded paths of the cells in this assay, it appears that the totalpath length for the neutrophils was not significantly affected by thetest compound. Rather, the compound affected neutrophil orientation orsense of direction, such that instead of migrating along the axis of thechemoattractant gradient, the cells migrated in an undirected or lessdirected manner.

E. Measurement of Bactericidal Capacity of Neutrophils

Given that the PI3Kδ inhibitor D-000 affects certain neutrophilfunctions detailed above, it was of interest to see whether the compoundaffects neutrophil-mediated bacterial killing. The effect of D-000 onneutrophil-mediated Staphylococcus aureus killing was studied accordingto the method described by Clark and Nauseef (pp. 7.23.4-7.23.6 in Vol.2, Supp. 6, Curr Protocols Immunol (Eds., Colligan et al.) (1994)).Purified human neutrophils (5×10⁶ cells/mL) (treated with either DMSO ora serial dilution of D-000 in DMSO) were mixed with autologous serum.Overnight-grown S. aureus cells were washed, resuspended in HBSS, andadded to the serum-opsonized neutrophils at a 10:1 ratio. Neutrophilswere allowed to internalize the bacteria by phagocytosis by incubationat 37° C. for 20 min. The noninternalized bacteria were killed by 10units/mL lysostaphin at 37° C. for 5 min and the total mixture wasrotated at 37° C. Samples were withdrawn at various times for up to 90min and the neutrophils were lysed by dilution in water. Viable bacteriawere counted by plating appropriate dilutions on trypticase-soy-agarplate and counting the S. aureus colonies after overnight growth.

As shown in FIG. 6, neutrophil-mediated killing of S. aureus was similarin samples treated with DMSO (control) and with D-000. These resultsindicate that the PI3Kδ inhibitor does not significantly affect theability of neutrophils to kill S. aureus, suggesting that PI3Kδ is notinvolved in this pathway of neutrophil function.

EXAMPLE 4

Characterization of Role of PI3Kδ in B Lymphocyte Function

The effects of the PI 3-kinase inhibitor on B cell functions includingclassical indices such as antibody production and specificstimulus-induced proliferation also were studied.

A. Preparation and Stimulation of B Cells from Peripheral Human Blood

Heparinized blood (200 mL) from healthy volunteers was mixed with anequal volume of D-PBS, layered on 10×10 mL FICOLL-PAQUE® (Pharmacia),and centrifuged at 1600 rpm for 30 min at room temperature. Peripheralblood mononuclear cells (PBMC) were collected from the FICOLL®/seruminterface, overlayed on 10 mL fetal bovine serum (FBS) and centrifugedat 800 rpm for 10 min to remove platelets. After washing, cells wereincubated with DYNAL® Antibody Mix (B cell kit) (Dynal Corp., LakeSuccess, N.Y.) for 20 min at 4-8° C. Following the removal of unboundantibody, PBL were mixed with anti-mouse IgG coated magnetic beads(Dynal) for 20 min at 4-8° C. with gentle shaking followed byelimination of labeled non-B cells on the magnetic bead separator. Thisprocedure was repeated once more. The B cells were resuspended inRPMI-1640 with 10% FBS, and kept on ice until further use.

B. Measurement of Antibody Production by Human B Cells

To study antibody production, B cells were aliquoted at 50-75×10³cells/well into 96-well plate with or without inhibitor, to which IL-2(100 U/mL) and PANSORBIN® (Calbiochem) Staphylococcus aureus cells(1:90,000) were added. Part of the media was removed after 24-36 hr, andfresh media (with or without inhibitor) and IL-2 were added. Cultureswere incubated at 37° C., in the presence of a CO, incubator foradditional 7 days. Samples from each condition (in triplicate) wereremoved, and analyzed for IgG and IgM, as measured by ELISA. Briefly,IMMULON® 4 96-well plates were coated (50 μL/well) with either 150 ng/mLdonkey antihuman IgG (H+L) (Jackson ImmunoResearch, West Grove Pa.), or2 μg/mL donkey antihuman IgG+IgM (H+L) (Jackson ImmunoResearch) inbicarbonate buffer, and left overnight at 4° C. After 3× washing withphosphate buffered saline containing 0.1% TWEEN®-80 (PBST) (350μL/well), and blocking with 3% goat serum in PBST (100 μL/well) for 1 hrat room temperature, samples (100 μL/well) of B cell spent media dilutedin PBST were added. For IgG plates the dilution range was 1:500 to1:10000, and for IgM 1:50 to 1:1000. After 1 hr, plates were exposed tobiotin-conjugated antihuman IgG (100 ng/mL) or antihuman IgM (200 ng/mL)(Jackson ImmunoResearch) for 30 min, following by streptavidin-HRP(1:20000) for 30 min, and finally, to TMB solution (1:100) with H₂O₂(1:10000) for 5 min, with 3×PBST washing between steps. Colordevelopment was stopped by H₂SO₄ solution, and plates were read on anELISA plate reader.

As shown in FIG. 7, D-000 significantly inhibited antibody production.IgM production was affected more than IgG production: half-maximalinhibition of IgM production was observed at about 1 μM, versus about 7μM for comparable inhibition of IgG production.

C. Measurement of B Cell Proliferation in Response to Cell Surface IgMStimulation

In the above experiment, the B cells were stimulated using PANSORBIN®.The effect of D-000 on B cell proliferation response when they werestimulated through their cell surface IgM using anti-IgM antibody alsowas measured. Murine splenocytes (Balb/c) were plated into 96-wellmicrotiter plates at 2×10⁵ cells per well in 10% FBS/RPMI. Appropriatedilutions of test inhibitor in complete medium were added to the cellsand the plates were incubated for 30-60 minutes prior to the addition ofstimulus. Following the preincubation with test inhibitor an F(ab′)₂preparation of goat antibody specific for the μ-chain of mouse IgM wasadded to the wells at a final concentration of 25 μg/mL. The plates wereincubated at 37° C. for 3 days and 1 μCi of [³H]-thymidine was added toeach well for the final four hours of culture. The plates were harvestedonto fiber filters washed and the incorporation of radiolabel wasdetermined using a beta counter (Matrix 96, Packard Instrument Co.,Downers Grove, Ill.) and expressed as counts per minute (CPM).

FIG. 8 shows the effect of D-000 on anti-IgM stimulated proliferation ofB cells. The compound inhibited anti-IgM-stimulated B cell proliferationin a dose-dependent manner. At about 1 μM, proliferation was reduced toits half-maximal value.

Because the compound D-000 inhibits B cell proliferation, it isenvisioned that this compound and other PI3Kδ inhibitors could be usedto suppress undesirable proliferation of B cells in clinical settings.For example, in B cell malignancy, B cells of various stages ofdifferentiation show unregulated proliferation. Based on the resultsshown above, one can infer that PI3Kδ selective inhibitors could be usedto control, limit, or inhibit growth of such cells.

EXAMPLE 5

Characterization of Role of PI3Kδ in T Lymphocyte Function

T cell proliferation in response to costimulation of CD3+CD28 wasmeasured. T cells were purified from healthy human blood by negativeselection using antibody coated magnetic beads according to themanufacturer's protocol (Dynal) and resuspended in RPMI. The cells weretreated with either DMSO or a serial dilution of D-000 in DMSO andplated at 1×10⁵ cells/well on a 96-well plate precoated with goatantimouse IgG. Mouse monoclonal anti-CD3 and anti-CD28 antibodies werethen added to each well at 0.2 ng/mL and 0.2 μg/mL, respectively. Theplate was incubated at 37° C. for 24 hr and [³H]-thymidine (1 μCi/well)was added. After another 18 hr incubation the cells were harvested withan automatic cell harvester, washed and the incorporated radioactivitywas quantified.

Although the PI3Kδ inhibitor D-000 inhibited anti-CD3- andanti-CD28-induced proliferation of T cells, its effect is not as strongas its effect on B cells or on some of the functions of neutrophils.Half-maximal inhibition of thymidine incorporation was not achieved atthe highest tested concentration, i.e., 10 μM D-000.

EXAMPLE 6

Characterization of Role of PI3Kδ in Osteoclast Function

To analyze the effect of the PI3Kδ inhibitor D-000 on osteoclasts, mousebone marrow cells were isolated and differentiated them to osteoclastsby treating the cells with Macrophage Colony Stimulating Factor⁻(mCSF⁻¹) and Osteoprotegerin Ligand (OPGL) in serum-containing medium(αMEM with 10% heat-inactivated FBS; Sigma) for 3 days. On day four,when the osteoclasts had developed, the medium was removed and cellswere harvested. The osteoclasts were plated on dentine slices at 10⁵cells/well in growth medium, i.e., αMEM containing 1% serum and 2% BSAwith 55 μg/mL OPGL and 10 ng/mL mCSF-1. After 3 hr, the medium waschanged to 1% serum and 1% BSA, with or without osteopontin (25 μg/mL)and the PI3K inhibitors (100 nM). The medium was changed every 24 hourswith fresh osteopontin and the inhibitors. At 72 hr, the medium wasremoved, and the dentine surfaces were washed with water to remove celldebris and stained with acid hematoxylin. Excess stain was washed andthe pit depths were quantitated using confocal microscopy.

As shown in Table 1, in two experiments, the PI 3-kinase inhibitors hadan inhibitory effect on osteoclast function. Both the nonspecificinhibitors LY294002 and wortmannin inhibited osteoclast activity.However, the PI3Kδ inhibitor D-000 had the most profound effect, as at100 nM this compound almost completely inhibited the osteoclastactivity.

TABLE 1 Osteopontin LY294002 + Wortmannin + (OPN) D-000 + OPN OPN OPN 10± 0.5 1 4.6 ± 0.22 5.7 ± 0.6  9 ± 0.4 1 5.8 ± 0.5    5 ± 0.5

EXAMPLE 7

Characterization of Role of PI3Kδ in Basophil Function

Assessment of the effect of a compound of the invention on basophilfunction was tested using a conventional histamine release assay,generally in accordance with the method described in Miura et al., JImmunol, 162:4198-206 (1999). Briefly, enriched basophils werepreincubated with test compounds at several concentrations from 0.1 nMto 1,000 nM, for 10 min at 37° C. Then, polyclonal goat antihuman IgE(0.1 μg/mL) or fMLP was added, and allowed to incubate for an additional30 min. Histamine released into the supernatant was measured using anautomated fluorometric technique. Two compounds were tested, shownbelow.

A dose-dependent decrease in histamine release was observed for3-(2-chlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-026) when the basophils were stimulated with anti-IgE. Thissuppression of histamine release was essentially 100% at 1,000 nM, withan EC₅₀ of about 25 nM. Another compound,3-(2-chlorophenyl)-2-(1H-pyrazolo[3,4-d]pyrimidin-4-ylsulfanylmethyl)-3H-quinazolin-4-one(D-999), in which the purine ring structure is rearranged, was lessefficacious in the inhibition of histamine release. Neither compoundelicited any effect when the basophils were stimulated with fMLP. Forcomparison, the nonselective PI3K inhibitor LY294002 was tested at 0.1nM and 10,000 nM, showing close to 100% inhibition of histamine releaseat the highest concentration.

These data indicate that inhibitors of PI 3-kinase delta activity can beused to suppress release of histamine, which is one of the mediators ofallergy. Since the activity of various PI 3-kinases are required forprotein trafficking, secretion, and exocytosis in many cell types, theabove data suggest that histamine release by other cells, such as mastcells, also can be disrupted by PI 3-kinase delta-selective inhibitors.

CHEMICAL SYNTHESIS EXAMPLES

Specific nonlimiting examples of compounds of the invention are providedbelow. It is understood in the art that protecting groups can beemployed where necessary in accordance with general principles ofsynthetic chemistry. These protecting groups are removed in the finalsteps of the synthesis under basic, acidic, or hydrogenolytic conditionsreadily apparent to those persons skilled in the art. By employingappropriate manipulation and protection of any chemical functionalities,synthesis of compounds of structural formula (I) not specifically setforth herein can be accomplished by methods analogous to the schemes setforth below.

Unless otherwise noted, all starting materials were obtained fromcommercial suppliers and used without further purification. Allreactions and chromatography fractions were analyzed by thin-layerchromatography (TLC) on 250 mm silica gel plates, visualized withultraviolet (UV) light or iodine (I₂) stain. Products and intermediateswere purified by flash chromatography or reverse-phase high performanceliquid chromatography.

The following abbreviations are used in the synthetic examples: aq(aqueous), H₂O (water), CHCl₃ (chloroform), HCl (hydrochloric acid),MeOH (methanol), NaOH (sodium hydroxide), NaOMe (sodium methoxide), TFA(trifluoroacetic acid), K₂CO₃ (potassium carbonate), SOCl₂ (thionylchloride), CH₂Cl₂ (methylene chloride), EtOAC (ethyl acetate), DMF(dimethylformamide), EtOH (ethanol), DMSO (dimethyl sulfoxide), NaHCO₃(sodium bicarbonate), TLC (thin layer chromatography), HPLC (highperformance liquid chromatography), HOBT (hydroxybenzotriazole), EDC(ethyldiethylaminopropylcarbodiimide), DIEA (diisopropylethylamine), andHOAc (acetic acid).

I. General Procedures

Procedure A

Thionyl chloride was added to a rapidly stirring solution of anthranilicacid or benzoic acid in benzene, and the mixture was stirred at refluxfor 5 to 18 hours. The reaction was concentrated in vacuo, and strippeddown twice with benzene. The resulting oil was dissolved in CHCl₃ and tothat solution was added the appropriate aniline. The reaction mixturewas heated to reflux and stirred until complete, as determined by TLC,at which point the reaction mixture was cooled to ambient temperature.The precipitate was removed by filtration, and the filtrate concentratedin vacuo. The crude product was purified by chromatography and/orrecrystallization from MeOH to provide amides 1a-1r.

Procedure B

To a rapidly stirring suspension of an amide in glacial acetic acid wasadded chloroacetyl chloride. The reaction mixture was heated to 120° C.,and allowed to stir at that temperature until complete, as determined byTLC. After brief cooling, the reaction mixture was concentrated invacuo. The crude residue was purified by extraction, chromatography,and/or recrystallization to provide chlorides 2a-2r.

Procedure C

A mixture of a chloride, either a nitrogen or a sulfur nucleophile, forexample, mercaptopurine monohydrate or adenine, and K₂CO₃ in DMF wasstirred at room temperature for 15-72 hours. The resulting suspensionwas poured into water, and kept at 4° C. for several hours. The crudesolid was filtered, washed with water, and purified by chromatography orrecrystallization to provide the final product.

EXAMPLE 8 Preparation of Intermediate Compounds: Amides2-Amino-N-(2-chlorophenyl)-4,5-dimethoxybenzamide (1a)

Prepared according to Procedure A using 4,5-dimethoxyanthranilic acid(5.0 g, 25.4 mmol) and SOCl₂ (5.5 mL, 76.1 mmol) in benzene (100 mL),followed by 2-chloroaniline (6.7 mL, 63.5 mmol) and CHCl₃ (75 mL). Theproduct was washed with aqueous NaHCO₃ (2×25 mL) and HCl (0.5 M, 75 mL)and purified by chromatography in CH₂Cl₂ to provide 4.3 g of a brownfoam (55%). ¹H NMR (CDCl₃) δ: 8.42 (dd, J=1.5, 8.3 Hz, 1H); 8.32 (br s,1H); 7.40 (dd, J=1.4, 8.0 Hz, 1H); 7.31 (dt, J=1.4, 7.9 Hz, 1H); 7.05(dt, J=1.5, 7.7 Hz, 1H); 7.03 (s, 1H); 6.24 (s, 1H); 3.88 (s, 3H); 3.87(s, 3H). MS (ES): m/z 307.0 (M+).

2-Amino-5-bromo-N-(2-chlorophenyl)benzamide (1b)

Prepared according to Procedure A using 2-amino-5-bromobenzoic acid (5.0g, 23.1 mmol) and SOCl₂ (7.0 mL, 95.9 mmol) in benzene (50 mL), followedby 2-chloroaniline (7.3 mL, 69.3 mmol) and CHCl₃ (50 mL). The productwas purified by two chromatographies in CH₂Cl₂ to provide 1.48 g of ayellow orange solid (20%). ¹H NMR (CDCl₂) δ: 8.36 (dd, J=1.2, 8.2 Hz,1H); 8.20 (br s, 1H); 7.62 (d, J=2.1 Hz, 1H); 7.42 (dd, J=1.3, 8.0 Hz,1H); 7.34 (dd, J=2.2, 8.8 Hz, 1H); 7.28-7.33 (m, 1H); 7.09 (dt, J=1.4,7.7 Hz, 1H); 6.62 (d, J=8.7 Hz, 1H); 5.57 (br s, 2H).

2-Amino-N-(2-chlorophenyl)-4-fluorobenzamide (1c)

Prepared according to Procedure A using 2-amino-4-fluorobenzoic acid(1.15 g, 7.41 mmol) and SOCl₂ (1.4 mL, 18.5 mmol) in benzene (25 mL),followed by 2-chloroaniline (1.6 mL, 14.8 mmol) and CHCl₃ (25 mL). Theproduct was chromatographed in CH₂Cl₂, then triturated from hexanes toprovide 1.02 g of an off-white solid (52%). ¹H NMR (CDCl₃) δ: 12.91 (brs, 1H); 8.72 (dd, J=2.7, 12 Hz, 1H); 8.34 (dd, J=6.4, 9.2 Hz, 1H); 8.29(dd, J=5.9, 8.8 Hz, 1H); 7.81 (dd, J=6.2, 8.8 Hz, 1H); 7.28 (dt, J=2.4,8.4 Hz, 1H); 7.21 (dd, J=2.4, 9.0 Hz, 1H); 6.92 (ddd, J=2.4, 7.3, 9.1Hz, 1H); 6.54 (ddd, J=2.4, 7.8, 8.8 Hz, 1H); 6.45 (dd, J=2.4, 11 Hz,1H); 5.93 (br s, 2H). MS (ES): m/z 265.0 (M+).

2-Amino-5-chloro-N-(2-chlorophenyl)benzamide (1d)

Prepared According to Procedure a Using 2-amino-5-chlorobenzoic acid(2.0 g, 11.7 mmol) and SOCl₂ (2.2 mL, 29.2 mmol) in benzene (50 mL),followed by 2-chloroaniline (2.5 mL, 23.3 mmol) and CHCl₃ (50 mL). Theproduct was purified by recrystallization from MeOH to provide 1.72 g ofa dark yellow solid (52%). ¹H NMR (CDCl₃) δ: 8.37 (dd, J=1.5, 8.3 Hz,1H); 8.22 (br s, 1H); 7.48 (d, J=2.3 Hz, 1H); 7.42 (dd, J=1.5, 8.1 Hz,1H); 7.31 (dt, J=1.4, 7.8 Hz, 1H); 7.22 (dd, J=2.4, 8.8 Hz, 1H); 7.09(dt, J=1.5, 7.7 Hz, 1H); 6.67 (d, J=8.8 Hz, 1H); 5.56 (br s, 2H).

2-Amino-N-(2-chlorophenyl)-6-fluorobenzamide (1e)

Prepared according to Procedure A using 2-amino-6-fluorobenzoic acid(2.0 g, 12.9 mmol) and SOCl₂ (2.3 mL, 32.2 mmol) in benzene (50 mL),followed by 2-chloroaniline (2.7 mL, 25.8 mmol) and CHCl₃ (50 mL). Theproduct was purified by chromatography in EtOAc/hexanes to provide 2.06g of a pale orange solid (60%). ¹H NMR (CDCl₃) δ: 9.00 (d, J=17 Hz, 1H);8.47 (d, J=8.3 Hz, 1H); 7.41 (d, J=8.0 Hz, 1H); 7.30 (t, J=7.9 Hz, 1H);7.10-7.20 (m, 1H); 7.07 (t, J=7.7 Hz, 1H); 6.49 (d, J=8.3 Hz, 1H); 6.03(br s, 2H). MS (ES): m/z 265.0 (M+).

2-Amino-6-chloro-N-(2-chlorophenyl)benzamide (1f)

Prepared according to Procedure A using 2-amino-6-chlorobenzoic acid(2.5 g, 14.6 mmol) and SOCl₂ (2.7 mL, 36.4 mmol) in benzene (75 mL),followed by 2-chloroaniline (3.1 mL, 29.1 mmol) and CHCl₃ (75 mL). Theproduct chromatographed in CH₂Cl₂ to provide 1.05 g of a yellow orangesolid (26%). ¹H NMR (CDCl₃) δ: 8.54 (d, J=8.1 Hz, 1H); 8.30 (br s, 1H);7.41 (dd, J=1.5, 8.0 Hz, 1H); 7.33 (t, J=7.8 Hz, 1H); 7.10 (t, J=8.1 Hz,1H); 7.09 (dt, J=1.6, 7.8 Hz, 1H); 6.78 (dd, J=0.4, 7.9 Hz, 1H); 6.63(dd, J=0.9, 8.2 Hz, 1H); 4.69 (br s, 2H). MS (ES): m/z 303.0 (M+22),281.0 (M+).

2-Amino-N-(2-chlorophenyl)-6-methylbenzamide (1g)

Prepared according to Procedure A using 2-amino-6-methylbenzoic acid(2.5 g, 16.5 mmol) and SOCl₂ (3.0 mL, 41.3 mmol) in benzene (75 mL),followed by 2-chloroaniline (3.5 mL, 33.0 mmol) and CHCl₃ (75 mL). Theproduct was chromatographed in CH₂Cl₂ to provide 2.19 g of a brown oil(51%). ¹H NMR (CDCl₃) δ: 8.58 (d, J=8.1 Hz, 1H); 7.99 (br s, 1H); 7.40(dd, J=1.4, 8.0 Hz, 1H); 7.34 (t, J=7.7 Hz, 1H); 7.11 (t, J=7.8 Hz, 1H);7.09 (dt, J=1.5, 7.7 Hz, 1H); 6.64 (d, J=7.5 Hz, 1H); 6.59 (d, J=8.1 Hz,1H); 4.29 (br s, 2H); 2.45 (s, 3H). MS (ES): m/z 283.0 (M+22).

2-Amino-3-chloro-N-(2-chlorophenyl)benzamide (1h)

Prepared according to Procedure A using 2-amino-3-chlorobenzoic acid(1.0 g, 5.82 mmol) and SOCl₂ (1.1 mL, 14.6 mmol) in benzene (25 mL),followed by 2-chloroaniline (1.2 mL, 11.7 mmol) and CHCl₃ (25 mL). Theproduct was recrystallized from MeOH to provide 1.29 g of a yellow solid(78%). ¹H NMR (CDCl₃) δ: 8.43 (dd, J=1.4, 8.3 Hz, 1H); 8.30 (br s, 1H);7.47 (dd, J=1.1, 8.0 Hz, 1H); 7.42 (d, J=8.0 Hz, 2H); 7.33 (dt, J=1.4,7.9 Hz, 1H); 7.09 (dt, J=1.5, 7.7 Hz, 1H); 6.68 (t, J=7.9 Hz, 1H); 6.13(br s, 2H). MS (ES): m/z 281.0 (M+).

2-Amino-N-biphenyl-2-yl-6-chlorobenzamide (1i)

Prepared according to Procedure A using 2-amino-6-chlorobenzoic acid(2.0 g, 11.7 mmol) and SOCl₂ (2.1 mL, 29.3 mmol) in benzene (60 mL),followed by 2-aminobiphenylamine (4.15 g, 24.5 mmol) and CHCl₃ (60 mL).The product was chromatographed in CH₂Cl₂ to provide 2.16 g of a foamydark-amber residue (57%). ¹H NMR (CDCl₃) δ: 8.48 (d, J=8.2 Hz, 1H); 7.79(br s, 1H); 7.34-7.46 (m, 6H); 7.20-7.30 (m, 2H); 7.00 (t, J=8.1 Hz,1H); 6.63 (dd, J=0.6, 7.9 Hz, 1H); 6.54 (d, J=8.3 Hz, 1H); 4.58 (br s,2H). MS (ES): m/z 323.1 (M+).

2-Amino-6-chloro-N-o-tolylbenzamide (1j)

Prepared according to Procedure A using 2-amino-6-chlorobenzoic acid(1.0 g, 5.83 mmol) and SOCl₂ (1.1 mL, 14.6 mmol) in benzene (30 mL),followed by o-toluidine (1.4 mL, 12.8 mmol) and CHCl₃ (30 mL). Theproduct was chromatographed in CH₂Cl₂ to provide 840 mg of an oilyyellow solid (55%). ¹H NMR (CDCl₃) δ: 7.96 (d, J=7.9 Hz, 1H); 7.60 (brs, 1H); 7.23-7.30 (m, 2H); 7.14 (t, J=7.5 Hz, 1H); 7.11 (t, J=8.3 Hz,1H); 6.78 (d, J=7.9 Hz, 1H); 6.64 (d, J=8.2 Hz, 1H); 4.73 (br s, 2H);2.35 (s, 3H). MS (ES): m/z 261.0 (M+).

2-Amino-6-chloro-N-(2-fluorophenyl)benzamide (1k)

Prepared according to Procedure Al using 2-amino-6-chlorobenzoic acid(2.0 g, 11.7 mmol) and SOCl₂ (2.1 mL, 29.1 mmol) in benzene (60 mL),followed by 2-fluoroaniline (2.3 mL, 23.4 mmol) and CHCl₃ (60 mL). Theproduct was chromatographed in CH₂Cl₂ to provide 1.05 g of a yellowsolid (34%). ¹H NMR (CDCl₃) δ: 8.45 (t, J=8.0 Hz, 1H); 8.01 (br s, 1H);7.02-7.22 (m, 4H); 6.78 (dd, J=0.5, 7.9 Hz, 1H); 6.64 (dd, J=0.8, 8.2Hz, 1H); 4.73 (br s, 2H). MS (ES): m/z 265.0 (M+).

2-Amino-6-chloro-N-(2-methoxyphenyl)benzamide (1l)

Prepared according to Procedure A using 2-amino-6-chlorobenzoic acid(2.0 g, 11.7 mmol) and SOCl₂ (2.1 mL, 29.1 mmol) in benzene (60 mL),followed by o-anisidine (2.6 mL, 23.4 mmol) and CHCl₃ (60 mL). Theproduct was chromatographed in CH₂Cl₂ to provide 2.61 g of a dark yellowoil (81%). ¹H NMR (CDCl₃) δ: 8.53 (dd, J=1.7, 7.9 Hz, 1H); 8.39 (br s,1H); 7.11 (dt, J=1.6, 7.8 Hz, 1H); 7.09 (t, J=8.1 Hz, 1H); 7.02 (dt,J=1.4, 7.8 Hz, 1H); 6.92 (dd, J=1.4, 8.0 Hz, 1H); 6.62 (dd, J=0.9, 8.2Hz, 1H); 4.66 (br s, 2H); 3.87 (s, 3H). MS (ES): m/z 277.0 (M+).

2-Amino-N-(2-chlorophenyl)-3-trifluoromethylbenzamide (1m)

Prepared according to Procedure A using 3-trifluoromethylanthranilicacid (2.0 g, 9.75 mmol) and SOCl₂ (1.8 mL, 24.4 mmol) in benzene (50mL), followed by 2-chloroaniline (2.1 mL, 19.5 mmol) and CHCl₃ (50 mL).The product was purified by recrystallization from MeOH to provide 2.38g yellow crystals (78%). ¹H NMR (CDCl₃) δ: 8.40 (dd, J=1.4, 8.3 Hz, 1H);8.25 (br s, 1H); 7.71 (d, J=7.8 Hz, 1H); 7.60 (d, J=7.8 Hz, 1H); 7.43(dd, J=1.4, 8.0 Hz, 1H); 7.34 (dt, J=1.3, 7.9 Hz, 1H); 7.11 (dt, J=1.5,7.7 Hz, 1H); 6.77 (t, J=7.8 Hz, 1H); 6.24 (br s, 2H). MS (ES): m/z 315.0(M+).

3-Aminonaphthalene-2-carboxylic acid (2-chlorophenyl)amide (1n)

Prepared according to Procedure A using 3-amino-2-napthoic acid (2.0 g,10.7 mmol) and SOCl₂ (1.9 mL, 26.7 mmol) in benzene (50 mL), followed by2-chloroaniline (2.3 mL, 21.4 mmol) and CHCl₃ (50 mL). The product wasrecrystallized from MeOH to provide 1.71 g of a brown solid (54%). ¹HNMR (CDCl₃) δ: 10.88 (br s, 1H); 9.21 (s, 1H); 8.91 (s, 1H); 8.70 (dd,J=1.0, 8.3 Hz, 1H); 7.95-8.01 (m, 1H); 7.87-7.94 (m, 1H); 7.60-7.68 (m,2H); 7.41 (dd, J=1.3, 8.0 Hz, 1H); 7.34 (dt, J=1.2, 7.8 Hz, 1H); 7.07(dt, J=1.4, 7.7 Hz, 1H). MS (ES): m/z 297.1 (M+).

2-Amino-N-(2-chlorophenyl)-4-nitrobenzamide (1o)

Prepared according to Procedure A using 4-nitroanthranilic acid (5.0 g,27.5 mmol) and SOCl₂ (5.0 mL, 68.6 mmol) in benzene (150 mL), followedby 2-chloroaniline (5.8 mL, 55.0 mmol) and CHCl₃ (150 mL). The productwas purified by chromatography in CH₂Cl₂ followed by recrystallizationfrom MeOH to provide 2.20 g of an orange-brown solid (31%). ¹H NMR(CDCl₃) δ: 8.41 (dd, J=1.3, 8.3 Hz, 1H); 8.31 (br s, 1H); 7.67 (d, J=8.6Hz, 1H); 7.57 (d, J=2.1 Hz, 1H); 7.52 (dd, J=2.2, 8.5 Hz, 1H); 7.44 (dd,J=1.3, 8.1 Hz, 1H); 7.35 (dt, J=1.3, 7.9 Hz, 1H); 7.13 (dt, J=1.4, 7.8Hz, 1H); 5.88 (br s, 2H). MS (ES): m/z 292.0 (M+).

2-Amino-N-(2-chlorophenyl)-5-hydroxybenzamide (1p)

Prepared according to Procedure A using 2-amino-5-hydroxybenzoic acid(5.0 g, 32.7 mmol) and SOCl₂ (6.0 mL, 81.6 mmol) in benzene (150 mL),followed by 2-chloroaniline (6.9 mL, 65.4 mmol) and CHCl₃ (150 mL). Theproduct was purified by two chromatographies in MeOH/CH₂Cl₂ to provide990 mg of a brown solid (12%). ¹H NMR (MeOH-d₄) δ: 7.92 (dd, J=1.6, 8.1Hz, 1H); 7.48 (dd, J=1.5, 7.7 Hz, 1H); 7.34 (dt, J=1.5, 7.7 Hz, 1H);7.20 (dt, J=1.7, 7.7 Hz, 1H); 7.16 (d, J=2.7 Hz, 1H); 6.83 (dd, J=2.7,8.7 Hz, 1H); 6.76 (d, J=8.7 Hz, 1H); [6.24 (br s, 2H)). MS (ES): m/z263.0 (M+).

2-Amino-N-(2-chlorophenyl)-4,5-difluorobenzamide (1q)

Prepared according to Procedure A using 4,5-difluoroanthranilic acid(2.0 g, 11.6 mmol) and SOCl₂ (2.1 mL, 28.9 mmol) in benzene (60 mL),followed by 2-chloroaniline (2.4 mL, 23.2 mmol) and CHCl₃ (60 mL). Theproduct was purified by two chromatographies in CH₂Cl₂ and EtOAc/hexanesto provide 769 mg of a yellow solid (23%). ¹H NMR (CDCl₃) δ: 8.69-8.82(m, 1H); 8.00 (dd, J=8.4, 9.0 Hz, 1H); 7.90 (dd, J=8.9, 12 Hz, 1H); 7.39(dd, J=6.8, 10 Hz, 1H); 6.53 (dd, J=6.6, 12 Hz, 1H); 6.41 (br s, 2H);5.79 (br s, 1H). MS (ES): m/z 283.1 (M+).

2-Amino-N-(2-chlorophenyl)-5-fluorobenzamide (1r)

Prepared according to Procedure A using 2-amino-5-fluorobenzoic acid(1.0 g, 6.45 mmol) and SOCl₂ (1.2 mL, 16.1 mmol) in benzene (30 mL),followed by 2-chloroaniline (1.4 mL, 12.9 mmol) and CHCl₃ (30 mL). Theproduct was triturated from CH₂Cl₂ to provide 985 mg of a mustard-yellowsolid (58%). ¹H NMR (CDCl₃) δ: 7.66 (dd, J=2.9, 8.7 Hz, 1H); 7.52-7.55(m, 1H); 7.32-7.37 (m, 3H); 7.09 (dt, J=3.0, 8.5 Hz, 1H); 6.71 (dd,J=4.3, 8.7 Hz, 1H). MS (ES): m/z 305.0 (M+40).

EXAMPLE 9 Preparation of Intermediate Compounds: Chlorides2-Chloromethyl-3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one (2a)

Prepared according to Procedure B with 1a (2.95 g, 9.63 mmol) andchloroacetyl chloride (2.3 mL, 28.9 mmol) in acetic acid (30 mL).Purified by extraction from aq. K₂CO₃ and recrystallization fromisopropanol to afford 1.61 g of a brown crystalline solid (46%). ¹H NMR(CDCl₃) δ: 7.59-7.66 (m, 2H); 7.45-7.56 (m, 3H); 7.20 (s, 1H); 4.37 (d,J=12 Hz, 1H), 4.08 (d, J=12 Hz, 1H); 4.04 (s, 3H); 4.00 (s, 3H). MS(ES): m/z 365.0 (M+).

6-Bromo-2-chloromethyl-3-(2-chlorophenyl)-3H-quinazolin-4-one (2b)

Prepared according to Procedure B with 1b (500 mg, 1.54 mmol) andchloroacetyl chloride (0.37 mL, 4.61 mmol) in acetic acid (10 mL).Purified by recrystallization from isopropanol to afford 490 mg of anoff-white solid (83%). ¹H NMR (CDCl₃) δ: 8.43 (d, J=2.3 Hz, 1H); 7.91(dd, J=2.3, 8.7 Hz, 1H); 7.67 (d, J=8.7 Hz, 1H); 7.60-7.65 (m, 1H);7.47-7.56 (m, 2H); 7.52 (t, J=5.3 Hz, 1H); 7.47-7.56 (m, 1H); 4.37 (d,J=12 Hz, 1H), 4.06 (d, J=12 Hz, 1H). MS (ES): m/z 385.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one (2c)

Prepared according to Procedure B with 1c (500 mg, 1.89 mmol) andchloroacetyl chloride (0.45 mL, 5.67 mmol) in acetic acid (10 mL).Purified by extraction from aqueous K₂CO₃ followed by recrystallizationfrom isopropanol to afford 501 mg of a yellow crystalline solid (82%).¹H NMR (CDCl₃) δ: 8.32 (dd, J=6.0, 8.9 Hz, 1H); 7.59-7.66 (m, 1H);7.50-7.55 (m, 3H); 7.44 (dd, J=2.4, 9.4 Hz, 1H); 7.27 (dt, J=2.5, 8.5Hz, 1H); 4.37 (d, J=12 Hz, 1H), 4.07 (d, J=12 Hz, 1H). MS (ES): m/z323.0 (M+).

6-Chloro-2-chloromethyl-3-(2-chlorophenyl)-3H-quinazolin-4-one (2d)

Prepared according to Procedure B with 1d (500 mg, 1.78 mmol) andchloroacetyl chloride (0.42 mL, 5.33 mmol) in acetic acid (10 mL).Purified by recrystallization from isopropanol to afford 555 mg of ayellow solid (92%). ¹H NMR (CDCl₃) δ: 8.27 (d, J=1.9 Hz, 1H); 7.74-7.78(m, 2H); 7.60-7.66 (m, 1H); 7.48-7.57 (m, 3H); 4.37 (d, J=12 Hz, 1H),4.07 (d, J=12 Hz, 1H). MS (ES): m/z 339.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one (2e)

Prepared according to Procedure B with 1e (500 mg, 1.89 mmol) andchloroacetyl chloride (0.45 mL, 5.67 mmol) in acetic acid (10 mL).Purified by extraction from aq. K₂CO₃ and recrystallization fromisopropanol to afford 430 mg of an off-white crystalline solid (70%). ¹HNMR (CDCl₃) δ: 7.76 (dt, J=5.3, 8.2 Hz, 1H); 7.56-7.65 (m, 2H);7.47-7.56 (m, 3H); 7.16-7.25 (m, 1H); 4.35 (d, J=12 Hz, 1H), 4.07 (d,J=12 Hz, 1H). MS (ES): m/z 323.0 (M+).

5-Chloro-2-chloromethyl-3-(2-chlorophenyl)-3H-quinazolin-4-one (2f)

Prepared according to Procedure B with if (1.00 g, 3.56 mmol) andchloroacetyl chloride (0.85 mL, 10.7 mmol) in acetic acid (15 mL).Purified by recrystallization from isopropanol to afford 791 mg of anoff-white crystalline solid (65%). ¹H NMR (CDCl₃) δ: 7.70 (s, 1H); 7.68(d, J=3.8 Hz, 1H); 7.61-7.65 (m, 1H); 7.55 (dd, J=2.7, 6.4 Hz, 1H); 7.51(d, J=3.1 Hz, 1H); 7.50 (s, 2H); 4.35 (d, J=12 Hz, 1H), 4.05 (d, J=12Hz, 1H). MS (ES): m/z 339.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one (2g)

Prepared according to Procedure B with 1g (2.18 g, 8.36 mmol) andchloroacetyl chloride (2.0 mL, 25.1 mmol) in acetic acid (40 mL).Purified by two chromatographies in CH₂Cl₂ and EtOAc/hexanes, followedby recrystallization from isopropanol to afford 638 mg of an off-whitecrystalline solid (24%). ¹H NMR (DMSO-d₆) δ: 7.73-7.80 (m, 3H);7.58-7.64 (m, 3H); 7.41 (d, J=7.4 Hz, 1H); 4.40 (d, J=12 Hz, 1H), 4.26(d, J=12 Hz, 1H); 2.74 (s, 3H). MS (ES): m/z 319.0 (M+).

8-Chloro-2-chloromethyl-3-(2-chlorophenyl)-3H-quinazolin-4-one (2h)

Prepared according to Procedure B with 1 h (500 mg, 1.78 mmol) andchloroacetyl chloride (0.49 mL, 6.13 mmol) in acetic acid (10 mL).Purified by extraction from aqueous K₂CO₃, followed by recrystallizationfrom isopropanol to afford 448 mg of a yellow solid (74%). ¹H NMR(CDCl₃) δ: 8.23 (dd, J=1.4, 8.0 Hz, 1H); 7.90 (dd, J=1.4, 7.8 Hz, 1H);7.61-7.66 (m, 1H); 7.51-7.55 (m, 3H); 7.47 (t, J=8.0 Hz, 1H); 4.48 (d,J=12 Hz, 1H), 4.12 (d, J=12 Hz, 1H). MS (ES): m/z 339.0 (M+).

3-Biphenyl-2-yl-5-chloro-2-chloromethyl-3H-quinazolin-4-one (21)

Prepared according to Procedure B with 1i (2.0 g, 6.20 mmol) andchloroacetyl chloride (1.5 mL, 18.6 mmol) in acetic acid (30 mL).Purified by chromatography in CH₂Cl₂, followed by recrystallization fromisopropanol to afford 1.44 g of an off-white solid (61%). ¹H NMR (CDCl₃)δ: 7.61-7.64 (m, 1H); 7.58-7.59 (m, 1H); 7.54-7.57 (m, 2H); 7.52-7.53(m, 1H); 7.45-7.52 (m, 2H); 7.24 (s, 5H); 3.92-4.03 (m, 2H). MS (ES):m/z 381.0 (M+).

5-Chloro-2-chloromethyl-3-o-tolyl-3H-quinazolin-4-one (2j)

Prepared according to Procedure B with 1j (750 mg, 2.88 mmol) andchloroacetyl chloride (0.69 mL, 8.63 mmol) in acetic acid (15 mL).Purified by chromatography in CH₂Cl₂, followed by recrystallization fromisopropanol to afford 340 mg of a white solid (37%). ¹H NMR (CDCl₃) δ:7.69 (d, J=2.1 Hz, 1H); 7.68 (q, J=7.4 Hz, 1H); 7.54 (dd, J=2.2, 7.0 Hz,1H); 7.35-7.47 (m, 3H); 7.21-7.25 (m, 1H); 4.27 (d, J=12 Hz, 1H); 4.11(d, J=12 Hz, 1H); 2.18 (s, 3H). MS (ES): m/z 319.0 (M+).

5-Chloro-2-chloromethyl-3-(2-fluorophenyl)-3H-quinazolin-4-one (2k)

Prepared according to Procedure B with 1k (1.0 g, 3.78 mmol) andchloroacetyl chloride (0.90 mL, 11.3 mmol) in acetic acid (20 mL).Purified by chromatography in CH₂Cl₂ to afford 484 mg of a pale pinksolid (40%). ¹H NMR (CDCl₃) δ: 7.69 (s, 1H); 7.68 (d, J=3.2 Hz, 1H);7.56 (d, J=3.0 Hz, 1H); 7.54 (d, J=3.0 Hz, 1H); 7.40-7.47 (m, 1H);7.35-7.38 (m, 1H); 7.27-7.32 (m, 1H); 4.35 (d, J=12 Hz, 1H); 4.18 (d,J=12 Hz, 1H). MS (ES): m/z 323.0 (M+).

5-Chloro-2-chloromethyl-3-(2-methoxyphenyl)-3H-quinazolin-4-one (21)

Prepared according to Procedure B with 1l (2.6 g, 9.41 mmol) andchloroacetyl chloride (2.2 mL, 28.2 mmol) in acetic acid (40 mL).Purified by chromatography in CH₂Cl₂, followed by recrystallization fromisopropanol to afford 874 mg of a pale yellow solid (28%). ¹H NMR(CDCl₃) δ: 7.55-7.74 (m, 2H); 7.47-7.54 (m, 2H); 7.34 (dd, J=1.7, 7.8Hz, 1H); 7.13 (dt, J=1.2, 7.7 Hz, 1H); 7.08 (dd, J=1.0, 8.4 Hz, 1H);4.29 (d, J=12 Hz, 1H); 4.11 (d, J=12 Hz, 1H); 3.80 (s, 3H). MS (ES): m/z335.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-8-trifluoromethyl-3H-quinazolin-4-one(2m)

Prepared according to Procedure B with 1m (500 mg, 1.59 mmol) andchloroacetyl chloride (0.38 mL, 4.77 mmol) in acetic acid (10 mL).Purified by recrystallization from isopropanol to afford 359 mg of awhite crystalline solid (61%). ¹H NMR (CDCl₃) δ: 8.51 (dd, J=1.0, 8.0Hz, 1H); 8.14 (d, J=7.3 Hz, 1H); 7.65 (dd, J=2.5, 5.6 Hz, 1H); 7.62 (d,J=3.9 Hz, 1H); 7.48-7.60 (m, 3H); 4.44 (d, J=12 Hz, 1H), 4.12 (d, J=12Hz, 1H). MS (ES): m/z 373.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-3H-benzo[g]quinazolin-4-one (2n)

Prepared according to Procedure B with 1n (500 mg, 1.68 mmol) andchloroacetyl chloride (0.40 mL, 5.05 mmol) in acetic acid (10 mL).Purified by chromatography in CH₂Cl₂ followed by recrystallization fromisopropanol to afford 232 mg of a light-brown solid (39%). ¹H NMR(CDCl₃) δ: 8.92 (s, 1H); 8.29 (s, 1H); 8.81 (d, J=8.3, 1H); 8.32 (d,J=8.3 Hz, 1H); 7.51-7.69 (m, 4H); 7.55 (d, J=5.2 Hz, 1H); 7.53 (d, J=3.8Hz, 1H); 4.43 (d, J=12 Hz, 1H), 4.12 (d, J=12 Hz, 1H). MS (ES): m/z355.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-7-nitro-3H-quinazolin-4-one (20)

Prepared according to Procedure B with 1o (500 mg, 1.71 mmol) andchloroacetyl chloride (0.41 mL, 5.14 mmol) in acetic acid (10 mL).Purified by extraction from aqueous K₂CO₃, followed by twochromatographies in CH₂Cl₂ to afford 338 mg of a yellow oil (56%). ¹HNMR (CDCl₃) δ: 8.64 (d, J=2.2 Hz, 1H); 8.48 (d, J=8.8 Hz, 1H); 8.32 (dd,J=2.2, 8.7 Hz, 1H); 7.66 (dd, J=2.5, 6.0 Hz, 1H); 7.52-7.59 (m, 3H);4.41 (d, J=12 Hz, 1H), 4.10 (d, J=12 Hz, 1H). MS (ES): m/z 350.0 (M+).

Acetic acid2-chloromethyl-3-(2-chlorophenyl)-4-oxo-3,4-dihydro-quinazolin-6-ylester (2p)

Prepared according to Procedure B with 1p (670 mg, 2.55 mmol) andchloroacetyl chloride (0.61 mL, 7.65 mmol) in acetic acid (10 mL).Purified by chromatography in 0-3% MeOH/CH₂Cl₂, followed byrecrystallization from isopropanol to afford 523 mg of the acetate aspale-peach crystals (57%). ¹H NMR (CDCl₃) δ: 8.00 (d, J=2.7 Hz, 1H);7.82 (d, J=8.8 Hz, 1H); 7.60-7.66 (m, 1H); 7.56 (dd, J=2.7, 8.8 Hz, 1H);7.51 (t, J=4.7 Hz, 2H); 7.50 (s, 1H); 4.38 (d, J=12 Hz, 1H), 4.08 (d,J=12 Hz, 1H); 2.36 (s, 3H). MS (ES): m/z 363.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-6,7-difluoro-3H-quinazolin-4-one (2q)

Prepared according to Procedure B with 1q (700 mg, 2.48 mmol) andchloroacetyl chloride (0.60 mL, 7.43 mmol) in acetic acid (12 mL).Purified by chromatography in CH₂Cl₂, followed by recrystallization fromisopropanol to afford 219 mg of a yellow crystalline solid (26%). ¹H NMR(CDCl₃) δ: 8.07 (dd, J=8.5, 9.7 Hz, 1H); 7.64 (dd, J=2.5, 5.6 Hz, 1H);7.60 (dd, J=3.5, 11 Hz, 1H); 7.55 (q, J=2.9 Hz, 3H); 7.52 (d, J=1.9 Hz,1H); 7.49-7.51 (m, 1H); 4.36 (d, J=12 Hz, 1H), 4.06 (d, J=12 Hz, 1H). MS(ES): m/z 341.0 (M+).

2-Chloromethyl-3-(2-chlorophenyl)-6-fluoro-3H-quinazolin-4-one (2r)

Prepared according to Procedure B with 1r (850 mg, 3.21 mmol) andchloroacetyl chloride (0.77 mL, 9.63 mmol) in acetic acid (15 mL).Purified by extraction from aqueous K₂CO₃, followed by chromatography inEtOAc/hexanes. A second chromatography in acetone/hexanes afforded 125mg of a white solid (12%). ¹H NMR (CDCl₃) δ: 7.95 (dd, J=2.9, 8.2 Hz,1H); 7.81 (dd, J=4.8, 9.0 Hz, 1H); 7.61-7.66 (m, 1H); 7.57 (dd, J=2.7,8.6 Hz, 1H); 7.57 (dd, J=2.7, 8.6 Hz, 1H); 7.52 (dd, J=3.2, 6.9 Hz, 1H);7.52 (br s, 2H); 4.38 (d, J=12 Hz, 1H), 4.08 (d, J=12 Hz, 1H). MS (ES):m/z 323.0 (M+).

EXAMPLE 10 Preparation of PI3Kδ Inhibitor Compounds Compound D-0012-(6-Aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2a (200 mg, 0.546mmol), adenine (81 mg, 0.601 mmol), K₂CO₃ (83 mg, 0.601 mmol), and DMF(4 mL). The crude product was recrystallized from ethanol (EtOH) toprovide 164 mg of a beige solid (65%), mp 281.5-282.7° C. (decomposes).¹H NMR (DMSO-d₆) δ: 8.06 (s, 1H); 8.04 (s, 1H); 7.76-7.81 (m, 1H);7.70-7.76 (m, 1H); 7.60-7.67 (m, 2H); 7.45 (s, 1H); 7.22 (s, 2H); 6.90(s, 1H); 5.08 (d, J=17 Hz, 1H); 4.91 (d, J=17 Hz, 1H); 3.87 (s, 3H);3.87 (s, 3H). ¹³C NMR (DMSO-d₆) ppm: 159.9, 156.2, 155.4, 152.9, 150.0,149.7, 149.4, 143.0, 141.9, 133.7, 132.1, 131.9, 131.2, 130.8, 129.3,118.4, 113.6, 108.4, 105.8, 56.5, 56.1, 44.7. MS (ES): m/z 464.1 (M+).Anal. calcd. for C₂₂H₁₈ClN₇O₃∘0.1C₂H₆O∘0.05KCl: C, 56.47; H, 3.97; Cl,7.88; N, 20.76. Found: C, 56.54; H, 4.05; Cl, 7.77; N, 20.55.

Compound D-0022-(6-Aminopurin-o-ylmethyl)-6-bromo-3-(2-chlorophenyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2b (100 mg, 0.260mmol), adenine (39 mg, 0.286 mmol), K₂CO₃ (40 mg, 0.286 mmol), and DMF(2 mL). The crude product was recrystallized from EtOH to provide 52 mgof an off-white solid (41%), mp 284.2-284.7° C. (decomposes). ¹H NMR(DMSO-d₆) δ: 8.24 (d, J=2.0 Hz, 1H); 8.05 (s, 1H); 8.03 (s, 1H); 7.98(dd, J=1.9, 8.6 Hz, 1H); 7.74-7.83 (m, 2H); 7.59-7.68 (m, 2H); 7.46 (d,J=8.7 Hz, 1H); 7.22 (s, 2H); 5.12 (d, J=17 Hz, 1H); 4.94 (d, J=17 Hz,1H). ¹³C NMR (DMSO-d₆) ppm: 159.5, 156.2, 152.9, 152.0, 150.1, 145.8,141.8, 138.4, 133.1, 132.2, 131.9, 131.1, 130.9, 130.1, 129.4, 128.9,122.4, 120.4, 118.4, 45.0. MS (ES): m/z 482.0 (M+). Anal. calcd. forC₂₀H₁₃ClBrN₇O.0.1KCl: C, 49.01; H, 2.67; Cl, 7.96; N, 20.00. Found: C,48.82; H, 2.82; Cl, 8.00; N, 19.79.

Compound D-0032-(6-Aminopurin-o-ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2c (100 mg, 0.310mmol), adenine (46 mg, 0.340 mmol), K₂CO₃ (47 mg, 0.340 mmol), and DMF(1 mL). The crude product was recrystallized from EtOH to provide 57 mgof a beige solid (44%), mp 216.8-217.2° C. ¹H NMR (DMSO-d₆) δ: 8.22 (dd,J=6.3, 8.7 Hz, 1H); 8.05 (s, 1H); 8.03 (s, 1H); 7.78-7.80 (m, 2H);7.61-7.64 (m, 2H); 7.46 (dt, J=2.1, 8.6 Hz, 1H); 7.32 (d, J=9.8 Hz, 1H);7.22 (s, 2H); 5.13 (d, J=17 Hz, 1H); 4.95 (d, J=17 Hz, 1H). ¹³C NMR(DMSO-d₆) ppm: 166.1 (d, J=253 Hz), 159.6, 155.8, 152.5, 149.7, 148.6(d, J=14 Hz), 141.4, 132.8, 131.8, 131.6, 130.8, 130.5, 129.8 (d, J=11Hz), 129.0, 118.1, 117.4, 116.2 (d, J=24 Hz), 112.7 (d, J=22 Hz), 44.6.MS (ES): m/z 422.0 (M+). Anal. calcd. for C₂₀H₁₃C1FN₇O.0.1H₂O (0.15KCl:C, 55.25; H, 3.06; Cl, 9.38; N, 22.55. Found: C, 55.13; H, 2.92; Cl,9.12; N, 22.30.

Compound D-0042-(6-Aminopurin-9-ylmethyl)-6-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2d (100 mg, 0.294mmol), adenine (44 mg, 0.323 mmol), K₂CO₃ (45 mg, 0.323 mmol), and DMF(1 mL). The crude product was recrystallized from EtOH to provide 50 mgof a yellow solid (39%), mp 294.5-294.8° C. (decomposes). ¹H NMR(DMSO-d₆) δ: 8.10 (d, J=2.2 Hz, 1H); 8.05 (s, 1H); 8.03 (s, 1H); 7.86(dd, J=2.4, 8.8 Hz, 1H); 7.75-7.82 (m, 2H); 7.59-7.67 (m, 2H); 7.53 (d,J=8.7 Hz, 1H); 7.22 (br s, 2H); 5.13 (d, J=17 Hz, 1H); 4.95 (d, J=17 Hz,1H). ¹³C NMR (DMSO-d₆) ppm: 159.7, 156.2, 152.9, 151.9, 150.1, 145.5,141.8, 135.7, 133.1, 132.3, 132.2, 131.9, 131.1, 130.9, 130.0, 129.4,125.9, 122.0, 118.4, 44.9. MS (ES): m/z 438.0 (M+). Anal. calcd. forC₂₀H₁₃Cl₂N₇O: C, 54.81; H, 2.99; N, 22.37. Found: C, 54.72; H, 2.87; N,22.18.

Compound D-0052-(6-Aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2e (200 mg, 0.619mmol), adenine (92 mg, 0.681 mmol), K₂CO₃ (94 mg, 0.680 mmol), and DMF(4 mL). The crude product was chromatographed in MeOH/CH₂Cl₂ to provide168 mg of an off-white solid (64%), mp 159-172° C. (graduallydecomposes). ¹H NMR (DMSO-d₆) δ: 8.10 (s, 1H); 8.08 (s, 1H); 7.73-7.89(m, 3H); 7.57-7.71 (m, 2H); 7.37-7.48 (m, 2H); 7.34 (d, J=11 Hz, 1H);7.30 (d, J=8.3 Hz, 1H); 5.14 (d, J=17 Hz, 1H); 4.94 (d, J=17 Hz, 1H).¹³C NMR (DMSO-d₆) ppm: 160.8 (d, J=264 Hz), 157.5 (d, J=4.2 Hz), 155.8,152.4, 152.4, 150.0, 148.7, 142.1, 136.4 (d, J=11 Hz), 133.0, 132.2,132.1, 131.2, 130.9, 129.4, 123.8 (d, J=3.6 Hz), 118.4, 114.5 (d, J=20Hz), 110.2 (d, J=6.0 Hz), 44.9. MS (ES): m/z 422.0 (M+). Anal. calcd.for C₂₀H₁₃ClFN₇O: C, 56.95; H, 3.11; Cl, 8.40; N, 23.24. Found: C,54.62; H, 3.32; Cl, 9.40; N, 21.29.

Compound D-0062-(6-Aminopurin-o-ylmethyl)-5-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2f (300 mg, 0.883mmol), adenine (131 mg, 0.972 mmol), K₂CO₃ (134 mg, 0.972 mmol), and DMF(4 mL). The crude product was chromatographed in MeOH/CH₂Cl₂ andrecrystallized from EtOH to provide 188 mg of a pale orange crystallinesolid (49%), mp 245.7-246.0© (starts to sweat at 220° C.). ¹H NMR(DMSO-d₆) δ: 8.06 (s, 1H); 8.04 (s, 1H); 7.76-7.81 (m, 2H); 7.72 (d,J=8.0 Hz, 1H); 7.59-7.66 (m, 3H); 7.41 (d, J=8.1 Hz, 1H); 7.26 (br s,2H); 5.11 (d, J=17 Hz, 1H); 4.93 (d, J=17 Hz, 1H). ¹³C NMR (DMSO-d₆)ppm: 158.5, 156.2, 152.9, 152.2, 150.1, 149.2, 141.8, 135.4, 133.3,133.2, 132.1, 132.0, 131.2, 130.9, 130.4, 129.4, 127.3, 118.4, 117.7,44.9. MS (ES): m/z 438.0 (M+). Anal. calcd. forC_(20H13)Cl₂N₂O.0.1C₂H₆O.0.05H₂O: C, 54.67; H, 3.11; Cl, 15.98; N,22.09. Found: C, 54.35; H, 3.00; Cl, 15.82; N, 22.31.

Compound D-0072-(6-Aminopurin-9-ylmethyl)-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2g (250 mg, 0.783mmol), adenine (116 mg, 0.862 mmol), K₂CO₃ (119 mg, 0.862 mmol), and DMF(4 mL). The crude product was recrystallized from EtOH to provide 93 mgof a pale yellow solid (28%), mp 190.7-190.9° C. ¹H NMR (DMSO-d₆) δ:8.05 (s, 1H); 8.03 (s, 1H); 7.76-7.79 (m, 1H); 7.71-7.74 (m, 1H);7.59-7.67 (m, 1H); 7.34 (d, J=7.4 Hz, 1H); 7.28 (d, J=8.2 Hz, 1H); 7.24(br s, 2H); 5.07 (d, J=17 Hz, 1H); 4.92 (d, J=17 Hz, 1H); 2.73 (s, 3H).¹³C NMR (DMSO-d₆) ppm: 161.1, 156.2, 152.8, 150.9, 150.1, 148.3, 141.9,141.0, 134.6, 133.6, 132.2, 131.9, 131.3, 130.8, 130.3, 129.3, 125.9,119.1, 118.4, 44.8, 22.8. MS (ES): m/z 418.1 (M+). Anal. calcd. forC₂H₁₆ClN₇O.H₂O: C, 57.87; H, 4.16; Cl, 8.13; N, 22.49. Found: C, 57.78;H, 3.99; Cl, 8.38; N, 22.32.

Compound D-0082-(6-Aminopurin-9-ylmethyl)-8-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2h (100 mg, 0.294mmol), adenine (44 mg, 0.324 mmol), K₂CO₃ (45 mg, 0.324 mmol), and DMF(1 mL). The crude product was chromatographed in MeOH/CH₂Cl₂ to provide50 mg of a pale yellow solid (39%), mp 273.3-273.5° C. (discolors). ¹HNMR (DMSO-d₆) δ: 8.11 (dd, J=1.3, 8.0 Hz, 1H); 8.08 (s, 1H); 8.05 (s,1H); 8.00 (dd, J=1.3, 7.8 Hz, 1H); 7.79-7.83 (m, 2H); 7.63-7.66 (m, 2H);7.56 (t, J=7.9 Hz, 1H); 7.21 (br s, 2H); 5.17 (d, J=17 Hz, 1H); 4.97 (d,J=17 Hz, 1H). ¹³C NMR (DMSO-d₆) ppm: 160.2, 156.1, 152.8, 152.2, 150.2,143.3, 142.0, 135.6, 133.1, 132.3, 131.9, 131.1, 131.0, 130.9, 129.4,128.4, 126.0, 122.5, 118.4, 45.0. MS (ES): m/z 438.0 (M+). Anal. calcd.for C₂₀H₁₃Cl₂N₇O.0.1CH₄O.0.6H₂O (0.15KCl: C, 52.09; H, 3.18; N, 21.15.Found: C, 51.85; H, 2.93; N, 21.01.

Compound D-0092-(6-Aminopurin-9-ylmethyl)-3-biphenyl-2-yl-5-chloro-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2i (400 mg, 1.05mmol), adenine (155 mg, 1.15 mmol), K₂CO₃ (159 mg, 1.15 mmol), and DMF(5 mL). The crude product was recrystallized from EtOH to provide 344 mgof a white solid (68%), mp 299.9-300.1° C. (discolors). ¹H NMR (DMSO-d₆)δ: 8.08 (s, 1H); 7.89 (s, 1H); 7.58-7.73 (m, 5H); 7.51 (d, J=7.9 Hz,1H); 7.46 (d, J=7.5 Hz, 2H); 7.27-7.41 (m, 3H); 7.14-7.27 (m, 3H); 5.14(d, J=17 Hz, 1H); 4.82 (d, J=17 Hz, 1H). ¹³C NMR (DMSO-d₆) ppm: 159.6,156.2, 152.8, 152.5, 150.0, 149.0, 141.7, 140.2, 137.7, 135.0, 133.3,133.2, 131.8, 130.7, 130.1, 129.8, 129.5, 128.8, 128.6, 128.4, 127.1,118.4, 117.6, 45.3. MS (ES): m/z 480.1 (M+). Anal. calcd. forC₂₆H₁₈ClN₇O: C, 65.07; H, 3.78; Cl, 7.39; N, 20.43. Found: C, 64.77; H,3.75; Cl, 7.43; N, 20.35.

Compound D-0105-Chloro-2-(9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2j (200 mg, 0.626mmol), 6-mercaptopurine monohydrate (93 mg, 0.546 mmol), K₂CO₃ (95 mg,0.689 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 125 mg of an off-white solid (46%), mp 213.9° C. ¹H NMR(DMSO-d₆) δ: 13.53 (br s, 1H); 8.49 (s, 1H); 8.44 (s, 1H); 7.78 (t,J=7.9 Hz, 1H); 7.63 (d, J=8.2 Hz, 1H); 7.59 (d, J=7.7 Hz, 1H); 7.49 (d,J=6.9 Hz, 1H); 7.24-7.41 (m, 3H); 4.32-4.45 (m, 2H); 2.14 (s, 3H). ¹³CNMR (DMSO-d₆) ppm: 158.9, 157.2, 154.2, 151.5, 149.7, 149.6, 143.5,136.1, 135.9, 135.1, 133.2, 131.3, 130.3, 130.0, 129.9, 129.1, 127.6,127.1, 117.8, 32.4, 17.5. MS (ES): m/z 438.0 (M+). Anal. calcd. forC₂₁H₁₅ClN₆OS: C, 58.00; H, 3.48; Cl, 8.15; N, 19.32; S, 7.37. Found: C,58.05; H, 3.38; Cl, 8.89; N, 18.38; S, 7.00.

Compound D-0115-Chloro-3-(2-fluorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2k (210 mg, 0.650mmol), 6-mercaptopurine monohydrate (122 mg, 0.715 mmol), K₂CO₃ (99 mg,0.715 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 240 mg of an off-white solid (84%), mp 244.0° C. ¹H NMR(DMSO-d₆) δ: 13.56 (br s, 1H); 8.50 (s, 1H); 8.45 (s, 1H); 7.81 (t,J=8.0 Hz, 1H); 7.74 (t, J=7.7 Hz, 1H); 7.67 (d, J=8.1 Hz, 1H); 7.62 (d,J=7.7 Hz, 1H); 7.46-7.55 (m, 1H); 7.29-7.42 (m, 2H); 4.47-4.59 (m, 2H).¹³C NMR (DMSO-d₆) ppm: 158.4, 157.3 (d, J=249 Hz), 156.4, 153.8, 151.0,149.1, 143.2, 135.0, 132.9, 131.8 (d, J=8.0 Hz), 130.8, 129.9, 126.7,125.3 (d, J=3.5 Hz), 123.6 (d, J=13 Hz), 117.0, 116.2 (d, J=19 Hz),31.7. MS (ES): m/z 439.0 (M+). Anal. calcd. for C₂₀H₁₂ClFN₆OS: C, 54.74;H, 2.76; Cl, 8.08; N, 19.15; S, 7.31. Found: C, 54.42; H, 2.88; Cl,8.08; N, 18.87; S, 7.08.

Compound D-0122-(6-Aminopurin-9-ylmethyl)-5-chloro-3-(2-fluorophenyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2k (210 mg, 0.650mmol), adenine (97 mg, 0.715 mmol), K₂CO₃ (99 mg, 0.715 mmol), and DMF(4 mL). The crude product was recrystallized from EtOH to provide 137 mgof a tan solid (50%), mp 295.6-295.8° C. (decomposes). ¹H NMR (DMSO-d₆)δ: 8.05 (s, 1H); 8.04 (s, 1H); 7.75 (t, J=7.6 Hz, 1H); 7.74 (t, J=7.9Hz, 1H); 7.62-7.69 (m, 1H); 7.61 (d, J=7.6 Hz, 1H); 7.47-7.55 (m, 1H);7.48 (d, J=7.8 Hz, 1H); 7.41 (d, J=8.0 Hz, 1H); 7.24 (br s, 2H); 5.19(d, J=17 Hz, 1H); 5.03 (d, J=17 Hz, 1H). ¹³C NMR (DMSO-d₆) ppm: 158.7,157.6 (d, J=250 Hz), 156.2, 152.8, 152.4, 150.0, 149.2, 141.8, 135.4,133.3, 132.5 (d, J=8.0 Hz), 131.0, 130.4, 127.3, 126.2 (d, J=3.5 Hz),123.1 (d, J=14 Hz), 118.4, 117.6, 117.2 (d, J=19 Hz), 45.1. MS (ES): m/z422.0 (M+). Anal. calcd. for C₂₀H₁₃ClFN₇O.0.05C₂H₆O: C, 56.92; H, 3.16;Cl, 8.36; N, 23.12. Found: C, 56.79; H, 3.20; Cl, 8.46; N, 22.79.

Compound D-0133-Biphenyl-2-yl-5-chloro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 21 (400 mg, 1.05mmol), 6-mercaptopurine monohydrate (196 mg, 1.15 mmol), K₂CO₃ (159 mg,1.15 mmol), and DMF (5 mL). The crude product was chromatographed inMeOH/CH₂Cl₂ and subsequently recrystallized from EtOH to provide 439 mgof a pale yellow crystalline solid (84%), mp 222.0-222.5° C. (dec). ¹HNMR (DMSO-d₆) δ: 13.56 (br s, 1H); 8.55 (s, 1H); 8.45 (s, 1H); 7.73 (t,J=8.0 Hz, 1H); 7.64 (d, J=7.7 Hz, 1H); 7.50-7.59 (m, 4H); 7.41-7.48 (m,1H); 7.25-7.38 (m, 5H); 4.41 (d, J=16 Hz, 1H); 4.16 (d, J=16 Hz, 1H).¹³C NMR (DMSO-d₆) ppm: 160.2, 157.0, 153.7, 151.5, 149.7, 149.3, 143.5,139.9, 137.8, 135.1, 134.1, 133.3, 131.5, 130.5, 130.3, 130.1, 129.1,128.9, 128.4, 128.4, 126.9, 117.5, 32.3. MS (ES): m/z 497.0 (M+). Anal.calcd. for C₂₆H₁₇ClN₆OS: C, 62.84; H, 3.45; Cl, 7.13; N, 16.91; S, 6.45.Found: C, 62.60; H, 3.47; Cl, 7.15; N, 16.65; S, 6.41.

Compound D-0145-Chloro-3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 21 (250 mg, 0.746mmol), 6-mercaptopurine monohydrate (140 mg, 0.821 mmol), K₂CO₃ (113 mg,0.821 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 254 mg of an off-white solid (76%), mp 237.0° C. (dec;discolors at 154.6° C.). ¹H NMR (DMSO-d₆) δ: 13.53 (br s, 1H); 8.52 (s,1H); 8.45 (s, 1H); 7.78 (t, J=7.9 Hz, 1H); 7.64 (d, J=8.0 Hz, 1H); 7.59(d, J=7.7 Hz, 1H); 7.48 (d, J=7.3 Hz, 1H); 7.42 (t, J=7.7 Hz, 1H); 7.15(d, J=8.2 Hz, 1H); 7.03 (t, J=7.5 Hz, 1H); 4.45 (s, 2H); 3.76 (s, 3H).¹³C NMR (DMSO-d₆) ppm: 158.9, 157.1, 154.8, 154.7, 151.5, 149.6, 143.6,135.1, 133.2, 131.3, 130.4, 130.0, 127.0, 124.8, 121.2, 117.8, 112.7,56.1, 32.0. MS (ES): m/z 451.0 (M+). Anal. calcd. forC₂₁H₁₅ClN₆O₂S.0.15C₂H₆O.0.05KCl: C, 55.43; H, 3.47; Cl, 8.07; N, 18.21;S, 6.95. Found: C, 55.49; H, 3.68; Cl, 7.95; N, 17.82; S, 6.82.

Compound D-0153-(2-Chlorophenyl)-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2e (200 mg, 0.619mmol), 6-mercaptopurine monohydrate (116 mg, 0.681 mmol), K₂CO₃ (94 mg,0.681 mmol), and DMF (5 mL). The crude product was recrystallized fromEtOH to provide 152 mg of a white solid (56%), mp 222.7-223.8° C.(discolors). ¹H NMR (DMSO-d₆) δ: 13.56 (br s, 1H); 8.48 (s, 1H); 8.44(s, 1H); 7.89 (dt, J=5.6, 8.1 Hz, 1H); 7.76 (dd, J=1.6, 7.3 Hz, 1H);7.67 (d, J=7.4 Hz, 1H); 7.56 (d, J=8.1 Hz, 1H); 7.47 (t, J=7.1 Hz, 1H),7.41-7.53 (m, 2H); 7.37 (dd, J=8.7, 11 Hz, 1H); 4.38-4.52 (m, 2H). ¹³CNMR (DMSO-d₆) ppm: 160.9 (d, J=264 Hz), 157.6, 156.8, 154.1, 151.5,149.6, 149.0, 143.6, 136.4 (d, J=11 Hz), 133.9, 132.2, 131.7, 131.6,130.5, 130.2, 128.8, 123.6, 114.4 (d, J=20 Hz), 110.2, 32.0. MS (ES):m/z 439.0 (M+). Anal. calcd. for C₂₀H₁₂ClFN₆OS.0.5C₂H₆O: C, 54.61; H,3.27; Cl, 7.68; N, 18.19; S, 6.94. Found: C, 54.37; H, 3.26; Cl, 7.89;N, 18.26; S, 6.55.

Compound D-0163-(2-Chlorophenyl)-6,7-dimethoxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2a (200 mg, 0.546mmol), 6-mercaptopurine monohydrate (102 mg, 0.601 mmol), K₂CO₃ (83 mg,0.601 mmol), and DMF (5 mL). The crude product was recrystallized fromEtOH to provide 172 mg of an off-white solid (65%), mp 160-180° C.(gradually decomposes). ¹H NMR (DMSO-d₆) δ: 13.55 (br s, 1H); 8.49 (s,1H); 8.44 (s, 1H); 7.72 (d, J=6.9 Hz, 1H); 7.66 (d, J=6.9 Hz, 1H)7.38-7.54 (m, 3H); 7.22 (s, 1H); 4.36-4.52 (m, 2H); 3.94 (s, 3H); 3.89(s, 3H). ¹³C NMR (DMSO-d₆) ppm: 160.1, 155.4, 151.5, 151.1, 149.4,143.2, 134.6, 132.3, 131.6, 131.5, 130.4, 128.7, 113.6, 108.4, 105.8,56.5, 56.1, 32.0. MS (ES): m/z 481.1 (M+). Anal. calcd. forC₂₂H₃₇ClN₆O₃S.0.5C₂H₆O.0.05KCl: C, 54.41; H, 3.97; Cl, 7.33; N, 16.55;S, 6.32. Found: C, 54.43; H, 3.94; Cl, 7.69; N, 16.69; S, 6.52.

Compound D-0176-Bromo-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2b (200 mg, 0.519mmol), 6-mercaptopurine monohydrate (97 mg, 0.570 mmol), K₂CO₃ (79 mg,0.572 mmol), and DMF (5 mL). The crude product was recrystallized fromEtOH to provide 123 mg of an off-white solid (47%), mp 212-242° C.(gradually decomposes). ¹H NMR (DMSO-d₆) δ: 13.07 (br s, 1H); 8.48 (s,1H); 8.44 (s, 1H); 8.24 (d, J=2.3 Hz, 1H); 8.06 (dd, J=2.3, 8.7 Hz, 1H);7.76 (dd, J=1.9, 7.4 Hz, 1H); 7.70 (d, J=8.7 Hz, 1H); 7.66 (d, J=8.1 Hz,1H); 7.51 (dd, J=2.1, 7.9 Hz, 1H); 7.46 (dd, J=1.9, 7.9 Hz, 1H); 4.47(s, 2H). ¹³H NMR (DMSO-d₆) ppm: 159.7, 156.8, 153.6, 151.5, 146.1,143.6, 138.5, 134.0, 132.1, 131.8, 131.5, 130.5, 130.2, 129.9, 128.9,128.8, 122.2, 120.3, 32.0. MS (ES): m/z 499.0 (M+). Anal. calcd. forC₂₀H₁₂ClBrN₆OS∘0.2C₂H₆O∘0.05KCl: C, 47.79; H, 2.59; N, 16.39; S, 6.25.Found: C, 47.56; H, 2.54; N, 16.25; S, 6.58.

Compound D-0183-(2-Chlorophenyl)-(9H-purin-6-ylsulfanylmethyl)trifluoromethyl-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2m (200 mg, 0.536mmol), 6-mercaptopurine monohydrate (100 mg, 0.588 mmol), K₂CO₃ (82 mg,0.593 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 148 mg of a white solid (56%), mp 218.5-219.4° C. ¹H NMR(DMSO-d₆) δ: 13.52 (br s, 1H); 8.48 (s, 1H); 8.44 (s, 1H); 8.43 (d,J=6.0 Hz, 1H); 8.26 (d, J=7.5 Hz, 1H); 7.84 (dd, J=2.5, 6.7 Hz, 1H);7.70-7.75 (m, 2H); 7.51-7.59 (m, 2H); 4.40-4.55 (m, 2H). ¹³C NMR(DMSO-d₆) ppm: 160.0, 157.2, 154.2, 151.4, 149.6, 144.4, 143.4, 133.8,133.0 (q, J=5.1 Hz), 132.0, 131.9, 131.6, 131.4, 130.6, 129.0, 127.3,125.2 (q, J=30 Hz), 123.6 (q, J=273 Hz), 121.8, 32.6. MS (ES): m/z 489.0(M+). Anal. calcd. for C₂₁H₁₂ClF₃N₆OS: C, 51.59; H, 2.47; Cl, 7.25; N,17.19; S, 6.56. Found: C, 51.51; H, 2.55; Cl, 7.37; N, 17.05; S, 6.38.

Compound D-0193-(2-Chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-benzo[g]quinazolin-4-one

Prepared according to Procedure C using Intermediate 2n (200 mg, 0.563mmol), 6-mercaptopurine monohydrate (105 mg, 0.619 mmol), K₂CO₃ (86 mg,0.619 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 128 mg of a dark yellow solid (48%), mp 247.8-254.4° C.(decomposes). ¹H NMR (DMSO-d₆) δ: 13.56 (br s, 1H); 8.90 (s, 1H); 8.50(s, 1H); 8.46 (s, 1H); 8.34 (s, 1H); 8.27 (d, J=8.2 Hz, 1H); 8.16 (d,J=8.2 Hz, 1H); 7.81 (dd, J=1.6, 7.3 Hz, 1H); 7.70 (t, J=7.5 Hz, 1H);7.61-7.74 (m, 2H); 7.49 (t, J=7.5 Hz, 1H); 7.44-7.53 (m, 1H); 4.44-4.56(m, 2H). ¹³C NMR (DMSO-d₆) ppm: 161.3, 151.6, 151.5, 143.9, 142.2,136.7, 134.4, 132.5, 131.8, 131.6, 130.5, 129.7, 129.3, 128.8, 128.6,128.3, 128.3, 127.1, 125.2, 119.5, 32.4. MS (ES): m/z 471.0 (M+). Anal.calcd. for C₂₄H₁₅ClN₆OS.0.2C₂H₆O.0.05KCl: C, 60.57; H, 3.37; Cl, 7.69;N, 17.37; S, 6.63. Found: C, 60.24; H, 3.46; Cl, 7.50; N, 17.34; S,6.69.

Compound D-0206-Chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2d (200 mg, 0.587mmol), 6-mercaptopurine monohydrate (110 mg, 0.646 mmol), K₂CO₃ (90 mg,0.651 mmol), and DMF (5 mL). The crude product was recrystallized fromEtOH to provide 113 mg of a yellow crystalline solid (42%), mp237.1-238.2° C. (decomposes). ¹H NMR (DMSO-d₆) δ: 13.55 (br s, 1H); 8.48(s, 1H); 8.44 (s, 1H); 8.11 (s, 1H); 7.94 (d, J=8.3 Hz, 1H); 7.78 (d,J=8.1 Hz, 2H); 7.66 (d, J=6.7 Hz, 1H); 7.48-7.56 (m, 2H); 4.48 (s, 2H).¹³C NMR (DMSO-d₆) ppm: 159.8, 156.8, 153.5, 151.5, 149.6, 145.8, 143.6,135.7, 134.0, 132.2, 132.1, 131.7, 131.5, 130.5, 130.2, 129.8, 128.8,125.8, 121.9, 32.0. MS (ES): m/z 455.0 (M+). Anal. calcd. forC₂₀H₁₂Cl₂N₆OS.0.1C₂H₆O.0.6H₂O (0.15KCl: C, 50.34; H, 2.89; Cl, 15.82; N,17.44; S, 6.65. Found: C, 50.02; H, 2.63; Cl, 15.51; N, 17.39; S, 6.81.

Compound D-0218-Chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2h (200 mg, 0.589mmol), 6-mercaptopurine monohydrate (124 mg, 0.726 mmol), K₂CO₃ (100 mg,0.726 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 202 mg of a white solid (75%), mp 211.9-212.7°(decomposes). ¹H NMR (DMSO-d₆) δ: 13.54 (br s, 1H); 8.47 (s, 1H); 8.44(s, 1H); 8.12 (d, J=7.9 Hz, 1H); 8.07 (d, J=7.6 Hz, 1H); 7.78 (d, J=7.5Hz, 1H); 7.67 (d, J=7.1 Hz, 1H); 7.58 (t, J=7.9 Hz, 1H); 7.42-7.54 (m,2H); 4.52 (s, 2H). ¹³C NMR (DMSO-dd ppm: 160.3, 156.9, 153.9, 151.5,149.7, 143.5, 135.7, 134.0, 132.1, 131.8, 131.4, 131.1, 130.5, 130.3,128.9, 128.3, 126.1, 122.4, 32.5. MS (ES): m/z 455.0 (M+). Anal. calcd.for C₂₀H₁₂Cl₂N₆OS: C, 52.76; H, 2.66; Cl, 15.57; N, 18.46; S, 7.04.Found: C, 52.65; H, 2.79; Cl, 15.32; N, 18.47; S, 7.18.

Compound D-0223-(2-Chlorophenyl)-7-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2c (200 mg, 0.619mmol), 6-mercaptopurine monohydrate (116 mg, 0.681 mmol), K₂CO₃ (95 mg,0.687 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 143 mg of a white crystalline solid (53%), mp151.4-154.2° C. (discolors). ¹H NMR (DMSO-d₆) δ: 13.55 (hr s, 1H); 8.48(s, 1H); 8.44 (s, 1H); 8.23 (dd, J=6.3, 8.7 Hz, 1H); 7.77 (dd, J=1.7,7.4 Hz, 1H); 7.64 (d, J=7.4 Hz, 1H); 7.57 (d, J=9.8 Hz, 1H); 7.45-7.52(m, 3H); 4.48 (s, 2H). ¹³C NMR (DMSO-d₆) ppm: 169.0 (d, J=253 Hz),162.6, 159.3, 157.0, 154.0, 152.2, 151.7 (d, J=13 Hz), 146.1, 136.5,134.7, 134.2, 134.0, 133.0, 132.6 (d, J=11 Hz), 131.3, 120.2, 118.9 (d,J=24 Hz), 115.3 (d, J=22 Hz), 34.6. MS (ES): m/z 439.0 (M+). Anal.calcd. for C₂₀H₁₂ClFN₆OS.0.4-C₂H₆O.0.4H₂O (0.15KCl: C, 52.52; H, 3.22;Cl, 8.57; N, 17.67. Found: C, 52.25; H, 3.11; Cl, 8.20; N, 17.69.

Compound D-0233-(2-Chlorophenyl)-7-nitro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2o (216 mg, 0.617mmol), 6-mercaptopurine monohydrate (116 mg, 0.681 mmol), K₂CO₃ (94 mg,0.680 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 212 mg of a yellow crystalline solid (74%), mp218.0-218.3° C. (decomposes). ¹H NMR (DMSO-d₆) δ: 13.56 (br s, 1H); 8.49(s, 1H); 8.42 (s, 1H); 8.38-8.45 (m, 2H); 8.31 (d, J=8.4 Hz, 1H); 7.81(d, J=6.5 Hz, 1H); 7.68 (d, J=6.7 Hz, 1H); 7.43-7.58 (m, 2H); 4.53 (s,2H). ¹³C NMR (DMSO-d₆) ppm: 157.7, 154.4, 153.3, 149.8, 149.3, 147.6,145.2, 141.4, 131.5, 129.8, 129.7, 129.2, 128.4, 127.1, 126.7, 122.7,120.3, 119.4, 29.9. MS (ES): m/z 466.0 (M+). Anal. calcd. forC₂₀H₁₂ClN₇O₃S.0.4C₂H₆O.0.05KCl: C, 51.19; H, 2.97; Cl, 7.63; N, 20.09;S, 6.57. Found: C, 51.27; H, 2.88; Cl, 7.40; N, 20.04; S, 6.52.

Compound D-0243-(2-Chlorophenyl)-6-hydroxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2p (200 mg, 0.552mmol), 6-mercaptopurine monohydrate (117 mg, 0.685 mmol), K₂CO₃ (95 mg,0.687 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 182 mg of a white solid, a mixture of the desiredproduct and the acetyl derivative. A portion of this material (120 mg)was suspended in a mixture of MeOH (2 mL) and aqueous NaHCO₃ (satd., 1mL) and stirred rapidly for 4 hours. The mixture was concentrated invacuo, suspended in H₂O (10 mL), and stored at 4° C. overnight. Thewhite solid was collected and dried to 103 mg (66%), mp 186-214° C.(gradually decomposes). ¹H NMR (DMSO-d₆) δ: 8.48 (s, 1H); 8.45 (s, 1H);7.71 (d, J=6.8 Hz, 1H); 7.62-7.64 (m, 2H); 7.43-7.51 (m, 2H); 7.40-7.43(m, 1H); 7.35 (d, J=8.8 Hz, 1H); 4.39-4.52 (m, 2H). ¹³C NMR (DMSO-d₆)ppm: 160.6, 157.1, 156.2, 151.4, 150.8, 149.3, 144.1, 140.2, 134.5,132.2, 131.6, 131.4, 130.4, 129.3, 128.7, 124.8, 121.7, 109.8, 32.0. MS(ES): m/z 437.0 (M+). Anal. calcd. for (2 C₂₀H₁₃ClN₆O₂S.0.1C₂H₆O.6H₂O:C, 49.68; H, 3.88; Cl, 7.26; N, 17.21; S, 6.57. Found: C, 49.43; H,3.62; Cl, 7.32; N, 17.07; S, 6.58.

Compound D-0255-Chloro-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2f (300 mg, 0.883mmol), 6-mercaptopurine monohydrate (165 mg, 0.972 mmol), K₂CO₃ (134 mg,0.972 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 341 mg of a pale orange crystalline solid (85%), mp233.7-234.4° C. (decomposes). ¹H NMR (DMSO-d₆) δ: 13.58 (br s, 1H); 8.50(s, 1H); 8.47 (s, 1H); 7.77-7.85 (m, 2H); 7.68 (d, J=8.1 Hz, 2H); 7.65(d, J=7.7 Hz, 1H); 7.41-7.56 (m, 2H); 4.45 (d, J=1.2 Hz, 2H). ¹³C NMR(DMSO-d₆) ppm: 158.7, 156.8, 153.8, 151.5, 149.6, 149.5, 143.5, 135.4,134.1, 133.3, 132.2, 131.6, 131.6, 130.5, 130.2, 128.8, 127.1, 117.6,32.0. MS (ES): m/z 455.0 (M+). Anal. calcd. forC₂₀H₁₂Cl₂N₆—OS.C₂H₆O.0.3H₂: C, 52.14; H, 3.70; Cl, 13.99; N, 16.58; S,6.33. Found: C, 52.07; H, 3.37; Cl, 13.40; N, 16.65; S, 6.42.

Compound D-0263-(2-Chlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2g (300 mg, 0.940mmol), 6-mercaptopurine monohydrate (176 mg, 1.03 mmol), K₂CO₃ (142 mg,1.03 mmol), and DMF (5 mL). The crude product was recrystallized fromEtOH to provide 324 mg of a white crystalline solid (79%), mp227.8-230.1° C. (decomposes). ¹H NMR (DMSO-d₆) δ: 13.57 (br s, 1H); 8.49(s, 1H); 8.47 (s, 1H); 7.69-7.78 (m, 2H); 7.66 (d, J=7.3 Hz, 1H); 7.55(d, J=7.9 Hz, 1H); 7.39-7.52 (m, 2H); 7.36 (d, J=6.9 Hz, 1H); 4.38-4.50(m, 2H); 2.74 (s, 3H). ¹³C NMR (DMSO-d₆) ppm: 161.2, 156.3, 152.4,151.5, 148.6, 143.9, 141.0, 134.6, 134.5, 132.3, 131.7, 131.4, 130.4,130.2, 128.7, 125.7, 119.0, 32.0, 22.8. MS (ES): m/z 435.0 (M+). Anal.calcd. for C₂₁H₁₅ClN₆OS.0.65C₂H₆O.0.1H₂O: C, 57.40; H, 4.13; Cl, 7.60;N, 18.01; S, 6.87. Found: C, 57.11; H, 3.96; Cl, 7.45; N, 17.79; S,6.90.

Compound D-0273-(2-Chlorophenyl)-6,7-difluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2q (200 mg, 0.586mmol), 6-mercaptopurine monohydrate (110 mg, 0.645 mmol), K₂CO₃ (89 mg,0.645 mmol), and DMF (4 mL). The crude product was recrystallized fromEtOH to provide 143 mg of a pale yellow crystalline solid (53%), mp207.8° C. (discolors; sweats at 136(C). ¹H NMR (DMSO-d₆) δ: 13.57 (br s,1H); 8.49 (s, 1H); 8.46 (s, 1H); 8.11 (t, J=9.4 Hz, 1H); 7.88 (dd,J=7.3, 11 Hz, 1H); 7.77 (dd, J=1.7, 7.3 Hz, 1H); 7.67 (d, J=7.4 Hz, 1H);7.42-7.55 (m, 2H); 4.48 (s, 2H). ¹³C NMR (DMSO-d₆) ppm: 159.5 (d, J=2.5Hz), 154.6 (dd, J=14, 255 Hz), 154.0 (d, J=1.5 Hz), 151.5, 149.3 (dd,J=14, 250 Hz), 145.1 (d, J=12 Hz), 143.9, 133.9, 132.1, 131.8, 131.4,130.5, 128.9, 118.0 (d, J=4.9 Hz), 115.8 (d, J=18 Hz), 114.6 (d, J=20Hz), 32.0. MS (ES): m/z 457.0 (M+). Anal. calcd. for C₂₀H₁₁ClF₂N₆OS: C,52.58; H, 2.43; Cl, 7.76; N, 18.40; S, 7.02. Found: C, 51.81; H, 2.37;Cl, 7.49; N, 18.04; S, 7.55.

Compound D-0283-(2-Chlorophenyl)-6-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Prepared according to Procedure C using Intermediate 2r (118 mg, 0.365mmol), 6-mercaptopurine monohydrate (68 mg, 0.402 mmol), K₂CO₃ (56 mg,0.402 mmol), and DMF (2 mL). The crude product was recrystallized fromEtOH to provide 103 mg of an off-white crystalline solid (64%), mp232.8-233.0° C. (discolors). ¹H NMR (DMSO-d₆) δ: 13.56 (br s, 1H); 8.48(s, 1H); 8.44 (s, 1H); 7.81-7.86 (m, 3H); 7.76 (d, J=7.5 Hz, 1H); 7.67(d, J=7.5 Hz, 1H); 7.40-7.54 (m, 2H); 4.48 (br s, 2H). ¹³C NMR (DMSO-d₆)ppm: 160.8 (d, J=247 Hz), 160.2 (d, J=3.3 Hz), 156.9, 152.3 (d, J=1.9Hz), 151.5, 149.7, 144.0, 143.6, 134.1, 132.1, 131.7, 131.5, 130.5,130.4, 130.2, 128.8, 124.0 (d, J=24 Hz), 122.0 (d, J=8.7 Hz), 111.7 (d,J=24 Hz), 32.0. MS (ES): m/z 439.0 (M+). Anal. calcd. forC₂₀H₁₂ClFN₆OS.0.2C₂H₆O.0.1H₂O: C, 54.46; H, 3.00; Cl, 7.88; N, 18.68.Found: C, 54.09; H, 2.73; Cl, 7.80; N, 18.77.

Compound D-0292-(6-Aminopurin-9-ylmethyl)-3-(2-isopropylphenyl)-5-methyl-3H-quinazolin-4-one

Thionyl chloride (2.2 mL, 30 mmol) was added to a stirred solution of2-amino-6-methylbenzoic acid (1.51 g, 10 mmol) in benzene (50 mL) andthe mixture was heated at reflux for 18 h. Once cooled, the solvent wasremoved in vacuo and stripped down twice with benzene (25 mL). Theresidue was dissolved in CHCl₃ (50 mL) and treated with2-isopropylaniline (2.83 mL, 20 mmol). The slurry was then heated atreflux for 3 h. At that time TLC (50% EtOAc/hexane) indicated that thereaction was complete. After cooling to room temperature, the reactionmixture was poured atop a 4 cm plug of silica gel and flushed throughwith 20% EtOAc/hexane. The product containing fractions were combinedand concentrated in vacuo. The residue was dissolved in HOAc (50 mL) andtreated with chloro-actyl chloride (1.6 mL, 20 mmol) and the mixture washeated at reflux for 18 h. The reaction was cooled and concentrated invacuo. The remaining HOAc was removed by azeotroping with toluene (25mL) three times. The residue was dissolved in toluene (10 mL) and pouredthrough a 4 cm plug of silica gel, flushing through with 20%EtOAc/hexane. The product containing fractions were identified by LCMS(MS (ES): m/z 327 (M+)), and concentrated in vacuo to afford 975 mg(30%) as a white foam. The white foam chloride (450 mg, 1.36 mmol) wasdissolved in DMF (10 mL) and treated with adenine (275 mg, 2.04 mmol)and K₂CO₃ (281 mg, 2.04 mmol) and the mixture was stirred overnight atroom temperature. The suspension was then poured into 200 mL of water,stirred at room temperature for 30 min then chilled in the refrigeratorfor 30 min. The resultant solid was collected by vacuum filtration andrecrystallized from EtOH to afford 285 mg (49%) of an off white solid.mp 258.0-258.2° C. ¹H NMR (DMSO-d₆) δ: 8.19 (s, 1H), 8.09 (s, 1H), 7.60(m, 3H), 7.45 (m, 2H), 7.23 (m, 3H), 5.11 (d, J=17.5 Hz, 1H), 4.71 (d,J=17.5 Hz, 1H), 2.68 (s, 3H), 2.73 (q, J=6.9 Hz, 1H), 1.34 (d, J=6.8 Hz,3H), 1.13 (d, J=6.8 Hz, 3H). ¹³C NMR (DMSO-d₆) ppm: 161.9, 156.2, 152.8,151.6, 150.1, 148.4, 146.1, 142.2, 140.8, 134.3, 133.7, 130.6, 130.0,129.0, 127.7, 127.6, 125.8, 119.2, 118.4, 44.8, 28.3, 24.4, 23.3, 22.9.MS (ES): m/z 426.4 (M+). Anal. calcd. for C₂₄H₂₃N₇O: C, 67.75; H, 5.45;N, 23.04. Found: C, 67.60; H, 5.45; N, 22.82.

Compound D-0302-(6-Aminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one

Thionyl chloride (2.2 mL, 30 mmol) was added to a stirred solution of2-amino-6-methylbenzoic acid (1.51 g, 10 mmol) in benzene (50 mL) andthe mixture was heated at reflux for 18 h. Once cooled, the solvent wasremoved in vacuo and stripped down twice with benzene (25 mL). Theresidue was dissolved in CHCl₃ (50 mL) and treated with o-toluidine(2.13 mL, 20 mmol). The slurry was then heated at reflux for 3 h. Atthat time TLC (50% EtOAc/hexane) indicated that the reaction wascomplete. After cooling to room temperature, the reaction mixture waspoured atop a 4 cm plug of silica gel and flushed through with 20%EtOAc/hexane. The product containing fractions were combined andconcentrated in vacuo. The residue was dissolved in HOAc (50 mL) andtreated with chloro-actyl chloride (1.6 mL, 20 mmol) and the mixture washeated at reflux for 18 h. The reaction was cooled and concentrated invacuo. The remaining HOAc was removed by azeotroping with toluene (25mL) three times. The residue was dissolved in toluene (10 mL) and pouredthrough a 4 cm plug of silica gel, flushing through with 20%EtOAc/hexane. The product containing fractions were identified by LCMS[MS (ES): m/z 299 (M+)), and concentrated in vacuo to afford 476 mg(16%) as a white foam. The white foam chloride (470 mg, 1.57 mmol) wasdissolved in DMF (10 mL) and treated with adenine (423 mg, 3.14 mmol)and K₂CO₃ (433 mg, 3.14 mmol) and the mixture was stirred overnight atroom temperature. The suspension was then poured into 200 mL of H₂O,stirred at room temperature for 30 min then chilled in the refrigeratorfor 30 min. The resultant solid was collected by vacuum filtration andrecrystallized from EtOH to afford 123 mg (20%) of an off white solid.mp 281.5-282.7° C. (decomposes). ¹H NMR (DMSO-d₆) δ: 8.07 (s, 1H); 8.05(s, 1H); 7.61 (t, J=7.8 Hz, 1H), 7.48 (m, 4H), 7.25 (m, 3H), 5.09 (d,J=17.4 Hz, 1H), 4.76 (d, J=17.4 Hz, 1H), 2.73 (s, 3H), 2.18 (s, 3H). ¹³CNMR (DMSO-d₆) ppm: 161.3, 156.2, 152.8, 151.4, 150.0, 148.5, 142.2,140.9, 136.1, 135.4, 134.3, 131.7, 130.1, 130.0, 129.0, 128.0, 125.8,119.2, 118.5, 44.8, 22.9, 17.4. MS (ES): m/z 398.2 (M+). Anal. calcd.for C₂₂H₁₉N₇O: C, 66.49; H, 4.82; N, 24.67. Found: C, 66.29; H, 4.78; N,24.72.

Compound D-0313-(2-Fluorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

Thionyl chloride (2.2 mL, 30 mmol) was added to a stirred solution of2-amino-6-methylbenzoic acid (1.51 g, 10 mmol) in benzene (50 mL) andthe mixture was heated at reflux for 18 h. Once cooled, the solvent wasremoved in vacuo and stripped down twice with benzene (25 mL). Theresidue was dissolved in CHCl₃ (50 mL) and treated with 2-fluoroaniline(1.93 mL, 20 mmol). The slurry was then heated at reflux for 3 h. Atthat time TLC (50% EtOAc/hexane) indicated that the reaction wascomplete. After cooling to room temperature, the reaction mixture waspoured atop a 4 cm plug of silica gel and flushed through with 20%EtOAc/hexane. The product containing fractions were combined andconcentrated in vacuo. The residue was dissolved in HOAc (50 mL) andtreated with chloro-actyl chloride (1.6 mL, 20 mmol) and the mixture washeated at reflux for 18 h. The reaction was cooled and concentrated invacuo. The remaining HOAc was removed by azeotroping with toluene (25mL) three times. The residue was dissolved in toluene (10 mL) and pouredthrough a 4 cm plug of silica gel, flushing through with 20%EtOAc/hexane. The product containing fractions were identified by LCMS[MS (ES): m/z 303 (M+)), and concentrated in vacuo to afford 1.12 g(37%) as a white foam. The white foam chloride (455 mg, 1.50 mmol) wasdissolved in DMF (10 mL) and treated with 6-mercaptopurine monohydrate(510 mg, 3.0 mmol) and K₂CO₃ (414 mg, 3.0 mmol) and the mixture wasstirred overnight at room temperature. The suspension was then pouredinto 200 mL of water, stirred at room temperature for 30 min thenchilled in the refrigerator for 30 min. The resultant solid wascollected by vacuum filtration and recrystallized from EtOH to afford487 mg (77%) of an off white solid. mp 151.9-152.2° C. ¹H NMR (DMSO-d₆)δ: 8.48 (s, 1H0, 8.44 (s, 1H), 7.70 (m, 2H), 7.48 (m, 2H), 7.33 (m, 3H),4.55 (d, J=15.1 Hz, 1H), 4.48 (d, J=15.1 Hz, 1H), 2.73 (s, 3H). ¹³C NMR(DMSO-d₆) ppm: 161.3, 157.8 (d, J=249.1 Hz), 156.9, 152.8, 151.5, 149.6,148.6, 143.6, 140.9, 134.7, 131.9 (d, J=8.0 Hz), 131.4, 130.2, 125.6 (d,J=3.6 Hz), 125.5, 124.4 (d, J=13.5 Hz), 118.8, 116.6 (d, J=19.6 Hz),56.4, 22.9. MS (ES): m/z 419.5 (M+). Anal. calcd. for C₂₁H₁₅FN₆OS∘0.15C₂H₆O: C, 60.14; H, 3.77; F, 4.47; N, 19.76; S, 7.54. Found: C, 59.89;H, 3.88; F, 4.42; N, 19.42; S, 7.23.

Compound D-0322-(6-Aminopurin-9-ylmethyl)-5-chloro-3-o-tolyl-3H-quinazolin-4-one

Prepared according to Procedure C using 2j (200 mg, 0.626 mmol), adenine(93 mg, 0.689 mmol), K₂CO₃ (95 mg, 0.689 mmol), and DMF (3 mL). Thecrude product was chromatographed in MeOH/CH₂Cl₂ to provide 101 mg of anoff-white solid (39%), mp 262.0-266.5° C. ¹H NMR (DMSO-d₆) δ: 8.08 (s,1H); 8.07 (s, 1H); 7.70 (t, J=8.0 Hz, 1H); 7.58 (dd, J=0.6, 7.9 Hz, 1H);7.43-7.57 (m, 4H); 7.36 (dd, J=0.7, 8.0 Hz, 1H); 7.26 (br s, 2H); 5.12(d, J=18 Hz, 1H); 4.78 (d, J=18 Hz, 1H); 2.20 (s, 3H). ¹³C NMR (DMSO-ddppm: 158.7, 156.2, 152.9, 152.7, 150.0, 149.4, 142.1, 136.1, 135.1,135.0, 133.2, 131.8, 130.3, 130.1, 128.9, 128.1, 127.2, 118.5, 117.9,44.9, 17.4. MS (ES): m/z 418.1 (M+). Anal. calcd. forC₂₁H₁₆ClN₇O∘0.1H₂O∘0.05KCl: C, 59.57; H, 3.86; Cl, 8.79; N, 23.16.Found: C, 59.65; H, 3.80; Cl, 8.70; N, 22.80.

Compound D-0332-(6-Aminopurin-9-ylmethyl)-5-chloro-3-(2-methoxyphenyl)-3H-quinazolin-4-one

Prepared according to Procedure C using 21 (250 mg, 0.746 mmol), adenine(111 mg, 0.821 mmol), K₂CO₃ (113 mg, 0.821 mmol), and DMF (4 mL). Thecrude product was chromatographed in MeOH/CH₂Cl₂ and recrystallized fromEtOH to provide 124 mg of a brown solid (38%), mp 257.0-257.1° C. ¹H NMR(DMSO-d₆) δ: 8.06 (s, 1H); 8.01 (s, 1H); 7.71 (t, J=8.0 Hz, 1H); 7.57(dd, J=0.9, 7.9 Hz, 1H); 7.52-7.59 (m, 1H); 7.50 (dd, J=1.6, 7.8 Hz,1H); 7.38 (dd, J=1.1, 8.2 Hz, 1H); 7.27 (dd, J=0.6, 8.3 Hz, 1H); 7.24(br s, 2H); 7.17 (dt, J=0.9, 7.6 Hz, 1H); 5.07 (d, J=17 Hz, 1H); 4.97(d, J=17 Hz, 1H); 3.79 (s, 3H). ¹³C NMR (DMSO-d₆) ppm: 158.8, 156.2,154.7, 153.2, 152.8, 150.1, 149.3, 142.0, 135.1, 133.2, 131.8, 130.1,130.1, 127.2, 123.8, 121.6, 118.4, 117.9, 113.1, 56.2, 44.8. MS (ES):m/z 434.0 (M+). Anal. calcd. for C₂₁H₁₆ClN₇O₂.0.5H₂O.0.04KCl: C, 56.57;H, 3.84; Cl, 8.27; N, 21.99. Found: C, 56.29; H, 3.75; Cl, 8.21; N,21.61.

The following compounds were made generally in accordance with theabove-described methods and serve to further illustrate specificembodiments of the compounds of the invention:

-   3-(2,6-dichlorophenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-034)-   3-(2-isopropylphenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-035)-   3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-036)-   3-benzyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one (D-037)-   3-butyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one (D-038)-   3-morpholin-4-yl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt (D-039)-   3-(3-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-040)-   3-(3-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-041)-   2-(9H-purin-6-ylsulfanylmethyl)-3-pyridin-4-yl-3H-quinazolin-4-one    (D-042)-   3-benzyl-5-fluoro-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-043)-   3-(4-methylpiperazin-1-yl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one,    acetate salt (D-044)-   [5-fluoro-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]acetic    acid ethyl ester (D-045)-   3-(2-methoxyphenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-046)-   3-(2-methoxyphenyl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-047)-   2-(6-aminopurin-9-ylmethyl)-3-(2-fluorophenyl)-5-methyl-3H-quinazolin-4-one    (D-048)-   2-(6-aminopurin-9-ylmethyl)-3-benzyl-5-fluoro-3H-quinazolin-4-one    (D-049)-   2-(6-aminopurin-9-ylmethyl)-3-butyl-3H-quinazolin-4-one (D-050)-   2-(6-aminopurin-9-ylmethyl)-3-morpholin-4-yl-3H-quinazolin-4-one,    acetate salt (D-051)-   3-(4-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one    (D-052).

Additional compounds of the present invention were prepared by thefollowing synthetic procedures.

The following intermediates were prepared by the above-describedProcedure A.

-   3a R=cyclopropyl-   3b R=cyclopropylmethyl-   3c R=phenethyl-   3d R=cyclopentyl-   3e R=3-(2-chloro)pyridyl-   3f R=4-(2-methyl)benzoic acid-   3g R=4-nitrobenzyl-   3h R=cyclohexyl-   3i R=E-(2-phenyl)cyclopropyl

Additional compounds of the present invention (D-053 through D-070)having the following core structure are discussed in the followingExperimental Section. All were prepared following Procedure C.

Core Structure:

Compound No. R R′ D-053 cyclopropyl C D-054 cyclopropylmethyl B D-055cyclopropylmethyl A D-056 cyclopropylmethyl C D-057 phenethyl B D-058phenethyl C D-059 cyclopentyl B D-060 cyclopentyl A D-0613-(2-chloro)pyridyl B D-062 3-(2-chloro)pyridyl A D-0634-(2-methyl)benzoic acid B D-064 cyclopropyl B D-065 cyclopropyl A D-0664-nitrobenzyl B D-067 cyclohexyl B D-068 cyclohexyl A D-069 cyclohexyl CD-070 E-(2-phenyl)cyclopropyl B

2-(2-Amino-9H-purin-6-ylsulfanylmethyl)-3-cyclopropyl-5-methyl-3H-quinazolin-4-one(D-053)

Prepared according to procedure C using 3a (100 mg, 0.4 mmol),2-amino-6-mercaptopurine (80 mg, 0.48 mmol), and K₂CO₃ (77 mg, 0.56mmol). The product was purified by trituration from H₂O. ¹H NMR(DMSO-d₆) δ: 7.89 (d, J=0.9 Hz, 1H); 7.54 (t, J=7.4 Hz, 1H); 7.34 (d,J=8.1 Hz, 1H); 7.19 (d, J=7.2 Hz, 1H); 6.28 (s, 2H); 4.94 (s, 2H); 2.70(s, 3H); 1.24 (d, J=6.5 Hz, 2H); 0.91 (s, 2H). MS (ES): m/z 380 (M+H),190.

3-Cyclopropylmethyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-054)

Prepared according to procedure C using 3b (300 mg, 1.14 mmol),6-mercaptopurine monohydrate (214 mg, 1.26 mmol), and K₂CO₃ (189 mg,1.37 mmol). The product was purified by trituration from H₂O, followedby recrystallization from MeOH. ¹H NMR (DMSO-d₆) δ: 13.60 (br s, 1H);8.72 (s, 1H); 8.48 (s, 1H); 7.63 (t, J=7.8 Hz, 1H); 7.42 (d, J=8.0 Hz,1H); 7.28 (d, J=7.3 Hz, 1H); 5.01 (s, 2H); 4.11 (d, J=6.8 Hz, 2H); 2.78(s, 3H); 1.35 (quint, J=6.2 Hz, 1H); 0.44-0.59 (m, 4H). MS (ES): m/z 379(M+H), 325.

2-(6-Aminopurin-9-ylmethyl)-3-cyclopropylmethyl-5-methyl-3H-quinazolin-4-one(D-055)

Prepared according to procedure C using 3b (300 mg, 1.14 mmol), adenine(170 mg, 1.26 mmol), and K₂CO₃ (189 mg, 1.37 mmol). The product waspurified by trituration from H₂O, followed by recrystallization fromMeOH. ¹H NMR (DMSO-d₆) δ: 8.21 (s, 1H); 8.10 (s, 1H); 7.52 (t, J=7.7 Hz,1H); 7.18-7.31 (m, 3H); 7.06 (d, J=8.1 Hz, 1H); 5.68 (s, 2H); 4.14 (d,J=6.8 Hz, 2H); 2.77 (s, 3H); 1.34 (quint, J=6.4 Hz, 1H); 0.45-0.60 (m,4H). MS (ES): m/z 362 (M+H), 308.

2-(2-Amino-9H-purin-6-ylsulfanylmethyl)-3-cyclopropylmethyl-5-methyl-3H-quinazolin-4-one(D-056)

Prepared according to procedure C using 3b (280 mg, 1.1 mmol),2-amino-6-mercaptopurine (200 mg, 1.2 mmol), and K₂CO₃ (180 mg, 1.3mmol). The product was purified by trituration from MeOH. NMR (DMSO-d₆)δ: 12.70 (br s, 1H); 7.95 (s, 1H); 7.64 (t, J=7.8 Hz, 1H); 7.44 (d,J=7.9 Hz, 1H); 7.28 (d, J=7.4 Hz, 1H); 6.41 (s, 2H); 4.91 (s, 2H); 4.05(d, J=6.8 Hz, 2H); 2.78 (s, 3H); 1.26-1.43 (m, 1H); 0.36-0.56 (m, 4H).MS (ES): m/z 394 (M+H), 340.

5-Methyl-3-phenethyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-057)

Prepared according to procedure C using 3c (750 mg, 2.4 mmol),6-mercaptopurine monohydrate (442 mg, 2.6 mmol), and K₂CO₃ (398 mg, 2.9mmol). The product was purified by trituration from H₂O. ¹H NMR(DMSO-d₆) δ: 13.61 (s, 1H); 8.71 (s, 1H); 8.48 (s, 1H); 7.65 (t, J=7.7Hz, 1H); 7.44 (d, J=7.9 Hz, 1H); 7.16-7.35 (m, 6H); 4.89 (s, 2H); 4.29(br t, J=7.9 Hz, 2H); 3.08 (br t, J=7.8 Hz, 2H); 2.81 (s, 3H). MS (ES):m/z 429 (M+H), 105.

2-(2-Amino-9H-purin-6-ylsulfanylmethyl)-5-methyl-3-phenethyl-3H-quinazolin-4-one(D-058)

Prepared according to procedure C using 3c (750 mg, 2.4 mmol),2-amino-6-mercaptopurine (435 mg, 2.6 mmol), and K₂CO₃ (398 mg, 2.9mmol). The product was purified by trituration from H₂O. ¹H NMR(DMSO-d₆) δ: 12.61 (s, 1H); 7.95 (s, 1H); 7.65 (t, J=7.7

Hz, 1H); 7.45 (d, J=7.9 Hz, 1H); 7.14-7.32 (m, 6H); 6.44 (s, 2H); 4.81(s, 2H); 4.24 (br t, J=7.9 Hz, 2H); 3.04 (br t, J=7.8 Hz, 2H); 2.81 (s,3H). MS (ES): m/z 444 (M+H), 340.

3-Cyclopentyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-059)

Prepared according to procedure C using 3d (100 mg, 0.36 mmol),6-mercaptopurine monohydrate (73 mg, 0.43 mmol), and K₂CO₃ (100 mg, 0.72mmol). The product was purified by recrystallization from MeOH. ¹H NMR(DMSO-d₆) δ: 13.62 (br s, 1H); 8.77 (s, 1H); 8.48 (s, 1H); 7.62 (t,J=7.7 Hz, 1H); 7.42 (d, J=7.8 Hz, 2H); 7.26 (d, J=7.4 Hz, 1H); 5.03 (s,2H); 4.80 (quint, J=8.0 Hz, 1H); 2.76 (s, 3H); 2.12-2.31 (m, 2H);1.79-2.04 (m, 4H); 1.44-1.58 (m, 2H). MS (ES): m/z 393 (M+H), 325.

2-(6-Aminopurin-9-ylmethyl)-3-cyclopentyl-5-methyl-3H-quinazolin-4-one(D-060)

Prepared according to procedure C using 3d (100 mg, 0.36 mmol), adenine(58 mg, 0.43 mmol), and K₂CO₃ (100 mg, 0.72 mmol). The product waspurified by recrystallization from MeOH. ¹H NMR (DMSO-d₆) δ: 8.15 (s,1H); 8.11 (s, 1H); 7.52 (t, J=7.7 Hz, 1H); 7.16-7.31 (m, 3H); 7.10 (d,J=8.0 Hz, 2H); 5.68 (s, 2H); 4.78 (quint, J=8.3 Hz, 1H); 2.74 (s, 3H);2.09-2.32 (m, 2H); 1.86-2.04 (m, 2H); 1.68-1.86 (m, 2H); 1.43-1.67 (m,2H). MS (ES): m/z 376 (M+H), 308, 154.

3-(2-Chloro-pyridin-3-yl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-061)

Prepared according to procedure C using 3e (500 mg, 1.6 mmol),6-mercaptopurine monohydrate (289 mg, 1.7 mmol), and K₂CO₃ (262 mg, 1.9mmol). The product was purified by trituration from H₂O. MS (ES): m/z436 (M+H), 200.

2-(6-Aminopurin-9-ylmethyl)-3-(2-chloro-pyridin-3-yl)-5-methyl-3H-quinazolin-4-one(D-062)

Prepared according to procedure C using 3e (500 mg, 1.6 mmol), adenine(230 mg, 1.7 mmol), and K₂CO₃ (262 mg, 1.9 mmol). The product waspurified by trituration from H₂O. ¹H NMR (DMSO-d₆) δ: 8.59 (dd, J=1.7,4.8 Hz, 1H); 8.22 (dd, J=1.7, 7.8 Hz, 1H): 8.025 (s, 1H); 8.017 (s, 1H);7.60-7.72 (m, 2H); 7.35 (t, J=8.2 Hz, 2H); 7.22 (s, 2H); 5.12 (d, J=17.0Hz, 1H); 5.02 (d, J=17.0 Hz, 1H); 2.72 (s, 3H). MS (ES): m/z 419 (M+H).

3-Methyl-4-[5-methyl-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]-benzoicacid (D-063)

Prepared according to procedure C using 3f (400 mg, 1.17 mmol),6-mercaptopurine monohydrate (219 mg, 1.29 mmol), and K₂CO₃ (226 mg,1.64 mmol). The product was purified by recrystallization from MeOH. ¹HNMR (DMSO-d₆) δ: 13.54 (br s, 1H); 8.44 (s, 1H); 8.42 (s, 1H); 7.80 (s,2H); 7.71 (t, J=7.7 Hz, 1H); 7.59 (d, J=8.6 Hz, 1H); 7.52 (d, J=7.9 Hz,1H); 7.34 (d, J=7.4 Hz, 1H); 4.46 (d, J=15.4 Hz, 1H); 4.34 (d, J=15.7Hz, 1H); 3.17 (d, J=4.4 Hz, 1H); 2.73 (s, 3H); 2.17 (s, 3H). MS (ES):m/z 459 (M+H).

3-Cyclopropyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-064)

Prepared according to procedure C using 3a (100 mg, 0.40 mmol),6-mercaptopurine monohydrate (90 mg, 0.53 mmol), and K₂CO₃ (97 mg, 0.7mmol). The product was purified by trituration from H₂O. ¹H NMR(DMSO-d₆) δ: 8.69 (d, J=0.8 Hz, 1H); 8.47 (s, 1H); 7.57 (d, J=7.9 Hz,1H); 7.37 (d, J=8.1 Hz, 1H); 7.23 (d, J=7.3 Hz, 1H); 5.08 (s, 2H);3.06-3.18 (m, 1H); 2.74 (s, 3H); 1.14-1.36 (m, 2H); 0.92-1.06 (m, 2H).

2-(6-Aminopurin-9-ylmethyl)-3-cyclopropyl-5-methyl-3H-quinazolin-4-one(D-065)

Prepared according to procedure C using 3a (100 mg, 0.40 mmol), adenine(94 mg, 0.7 mmol), and K₂CO₃ (121 mg, 0.88 mmol). The product waspurified by trituration from H₂O. ¹H NMR (DMSO-d₆) δ: 8.19 (d, J=0.9 Hz,1H); 8.09 (d, J=1.0 Hz, 1H); 7.48 (t, J=7.8 Hz, 1H); 7.13-7.29 (m, 3H);7.04 (d, J=8.1 Hz, 1H); 5.74 (s, 2H); 3.00-3.13 (m, 1H); 2.73 (s, 3H);1.18-1.38 (m, 2H); 0.94-1.09 (m, 2H).

5-Methyl-3-(4-nitro-benzyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-066)

Prepared according to procedure C using 3g (200 mg, 0.58 mmol),6-mercaptopurine monohydrate (148 mg, 0.87 mmol), and K₂CO₃ (160 mg,1.16 mmol). The product was purified by trituration from MeOH. ¹H NMR(DMSO-d₆) δ: 13.44 (br s, 1H); 8.50 (s, 1H); 8.31 (s, 1H); 8.03 (d,J=8.6 Hz, 2H); 7.58 (t, J=7.9 Hz, 1H); 7.37 (d, J=8.3 Hz, 3H); 7.22 (d,J=7.5 Hz, 1H); 5.44 (s, 2H); 4.70 (s, 2H); 2.66 (s, 3H). MS (ES): m/z460 (M+H).

3-Cyclohexyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-067)

Prepared according to procedure C using 3h (150 mg, 0.52 mmol),6-mercaptopurine monohydrate (97 mg, 0.57 mmol), and K₂CO₃ (86 mg, 0.62mmol). The product was purified by trituration from MeOH. ¹H NMR(DMSO-d₆) δ: 13.66 (br s, 1H); 8.82 (s, 1H); 8.50 (s, 1H); 7.62 (t,J=7.7 Hz, 1H); 7.42 (d, J=8.0 Hz, 1H); 7.26 (d, J=7.3 Hz, 1H); 5.01 (s,2H); 4.11 (br s, 1H); 2.75 (s, 3H); 2.38-2.65 (m, 2H); 1.58-1.90 (m,4H); 1.37-1.57 (m, 1H); 0.71-1.26 (m, 3H). MS (ES): m/z 407 (M+H), 325.

2-(6-Aminopurin-9-ylmethyl)-3-cyclohexyl-5-methyl-3H-quinazolin-4-one(D-068)

Prepared according to procedure C using 3h (150 mg, 0.52 mmol), adenine(77 mg, 0.57 mmol), and K₂CO₃ (86 mg, 0.62 mmol). The product waspurified by trituration from MeOH. ¹H NMR (DMSO-d₆) δ: 8.15 (s, 2H);7.54 (t, J=7.9 Hz, 1H); 7.06-7.35 (m, 4H); 5.65 (s, 2H); 4.09 (br s,1H); 2.73 (s, 3H); 1.41-1.90 (m, 6H); 0.99-1.34 (m, 4H). MS (ES): m/z390 (M+H), 308.

2-(2-Amino-9H-purin-6-ylsulfanylmethyl)-3-cyclohexyl-5-methyl-3H-quinazolin-4-one(D-069)

Prepared according to procedure C using 3h (150 mg, 0.52 mmol),2-amino-6-mercaptopurine (95 mg, 0.57 mmol), and K₂CO₃ (86 mg, 0.62mmol). The product was purified by reversed-phase HPLC (C18 Luna column,4.6×250 mm, 4.7 mL/min, 10-75% acetonitrile/water over 15 min, 100%acetonitrile at 18 min, detector at 220). MS (ES): m/z 422 (M+H), 340,170.

5-Methyl-3-(E-2-phenyl-cyclopropyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-070)

Prepared according to procedure C using 3i and 6-mercaptopurinemonohydrate). The product was purified by reversed-phase HPLC (C18 Lunacolumn, 4.6×250 mm, 4.7 mL/min, 10-75% acetonitrile/water over 15 min,100% acetonitrile at 18 min, detector at 220). MS (ES): m/z 441.

The following Methods 1 and 2 were used as the HPLC analyses for thefollowing compounds:

HPLC Method 1. Column: 2×50 mm C18 Luna column (from Phenomenex), flowrate: 0.3 mL/min, UV detection at 214 and 254 nm. Initial conditions: 2%solvent B in solvent A; t=3 min, 20% Solvent B; t=6 min, 80% Solvent B,where Solvent A=water with 0.05% formic acid and Solvent B=acetonitrilewith 0.05% formic acid.

HPLC Method 2. Column: 2×50 mm C18

Luna column (from Phenomenex), flow rate: 0.3 mL/min, UV detection at214 and 254 nm. Initial conditions: 10-100% solvent B in solvent A over6 min, where Solvent A=water and Solvent B=acetonitrile.

Compounds D-070A and D-070B were prepared as follows:

2-(6-Aminopurin-9-ylmethyl)-3-(2-benzyloxyphenyl)-5-methyl-3H-quinazolin-4-one(D-070A)

Using procedure A, 6-methylanthranilic acid and 2-benzyloxyaniline wereconverted to Intermediate (1s). Using procedure B, Intermediate (1s) wasconverted to Intermediate (2s). Using procedure C, Intermediate (2s) wasconverted to D-070A. Retention time using HPLC Method 2: 4.7 min. LRMS(ES pos.) m/z=490 (M+1).

2-(6-Aminopurin-9-ylmethyl)-3-(2-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one(D-070B)

A mixture of D-070A (35 mg, 0.07 mmol) and Pd(OH)₂ (20% by wt. on C, 50mg) in ethanol (5 mL) was shaken 24 hours under 40 psi of hydrogen gas.The catalyst was removed by filtration through a 0.22 um celluloseacetate membrane (Corning), and the filtrate was concentrated in vacuoto afford the solid product (10 mg). Retention time using HPLC Method 2:3.6 min. LRMS (ES pos.) m/z=400 (M+1).

Compounds D-070C through D-070F were prepared according to the followingscheme.

2-(6-Aminopurin-9-ylmethyl)-5-methyl-3-{2-(2-(1-methylpyrrolidin-2-yl)-ethoxy)-phenyl}-3H-quinazolin-4-one(D-070C)

A mixture of D-070B (30 mg, 0.075 mmol),2-(2-chloroethyl)-1-methylpyrrolidine hydrochloride (28 mg, 0.15 mmol),and potassium carbonate (50 mg, 0.36 mmol) in DMF (0.3 mL) was stirredat 80° C. for 16 hours. The solvent was removed in vacuo, then theresidue was dissolved in DMSO/water (1 mL) and purified by HPLC in twoportions (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 2-50%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, 0.05% TFAin all solvents, detector at 2201). Appropriate fractions wereconcentrated in vacuo, then concentrated twice from 1 N HCl (1 mL) toyield the final product as the HCl salt (10 mg). Retention time usingHPLC method 1: 4.7 min. LRMS (ES pos.) m/z=511 (M+1).

2-(6-Aminopurin-9-ylmethyl)-3-(2-(3-dimethylaminopropoxy)-phenyl)-5-methyl-3H-quinazolin-4-one(D-070D)

A mixture of D-070B (30 mg, 0.075 mmol),2-chloroethyl)-1-methylpyrrolidine hydrochloride (28 mg, 0.15 mmol), andpotassium carbonate (50 mg, 0.36 mmol) in DMF (0.3 mL) was stirred at80° C. for 16 h. The solvent was removed in vacuo, then the residue wasdissolved in DMSO/water (1 mL) and purified by HPLC in two portions (C18Luna column, 4.6×250 mm, 4.7 mL/min, 2-50% acetonitrile/water over 15min, 100% acetonitrile at 18 min, 0.05% TFA in all solvents, detector at2201). Appropriate fractions were concentrated in vacuo, thenconcentrated twice from 1 N HCl (1 mL) to yield the final product as theHCl salt (14 mg). Retention time using HPLC Method 1: 4.5 min. LRMS (ESpos.) m/z=485 (M+1).

2-(6-Aminopurin-9-ylmethyl)-5-methyl-3-(2-prop-2-ynyloxyphenyl)-3H-quinazolin-4-one(D-070E)

A mixture of D-070B (20 mg, 0.05 mmol), propargyl chloride (0.025 mL,0.33 mmol), and potassium carbonate (14 mg, 0.1 mmol) in DMF (0.3 mL)was stirred at 80° C. for 16 hours. The reaction mixture was cooled toroom temperature, treated with water (5 mL), and the resulting darkbrown precipitate was collected by filtration. The crude solid wasdissolved in 0.6 mL DMSO and purified by HPLC (C18 Luna column, 4.6×250mm, 4.7 mL/min, 10-75% acetonitrile/water over 15 min, 100% acetonitrileat 18 min, detector at 2201). Appropriate fractions were concentrated invacuo to yield the final product as a white solid (4 mg.). Retentiontime using HPLC Method 2: 4.1 min. LRMS (ES pos.) m/z=438 (M+1).

2-{2-(2-(6-Aminopurin-9-ylmethyl)-5-methyl-4-oxo-4H-quinazolin-3-yl)-phenoxy}-acetamide(D-070F)

A mixture of D-070B (20 mg, 0.05 mmol), 2-chloroacetamide (14 mg, 0.15mmol), and potassium carbonate (21 mg, 0.15 mmol) in DMF (0.3 mL) wasstirred at 80° C. for 16 hours. The reaction mixture was cooled to roomtemperature, treated with water (5 mL), and the resulting precipitatewas collected by filtration. Retention time using HPLC Method 2: 3.4min. LRMS (ES pos.) m/z=457 (M+1).

Additional compounds of the present invention were prepared by thefollowing synthetic procedures.

Additional compounds of the invention follow, together with thesynthetic route to compounds D-071 to D-118.

Procedure D

Procedure E

Procedure F

Procedure D: A mixture of amide 4a or 4b, FMOC-glycyl-chloride, andglacial acetic acid was heated to 120° C. for 1 to 4 hours. Theresulting mixture was concentrated in vacuo and purified by flashchromatography to provide the protected, cyclized amine. This materialwas combined with 10 equivalents octanethiol and a catalytic amount ofDBU in THF and stirred at ambient temp until consumption of startingmaterial was indicated by LCMS. The reaction was poured directly onto aflash column (equilibrated in CH₂Cl₂) and eluted with 0-5% MeOH/CH₂Cl₂to provide the free amine, 5a or 5b. Compound 5c was prepared in ananalogous manner using (±) FMOC-alanyl-chloride in place of FMOC-glycylchloride.

Procedure D1: Amide 4a was admixed with Cbz-α-aminobutyric acid OSu,DIEA, DMAP (cat.), and toluene, then stirred at reflux for 3 days. Theresulting mixture was purified by flash chromatography (EtOAc/hexanes)to provide the protected, cyclized amine. The amine was dissolved inEtOH with a catalytic amount of Pd/C, and allowed to stir at ambienttemperature under H₂ until a complete reaction was indicated by LCMS.The reaction mixture was filtered, and the filtrate concentrated invacuo. Purification by flash chromatography (MeOH/CH₂Cl₂) provided thefree amine 5d.

Procedure D2: Amide 4a was combined with Boc-Serine(OBn)-OSu, DIEA, DMAP(cat.), and toluene, then stirred at reflux for 4 days. The resultingmixture was purified by flash chromatography (EtOAc/hexanes) to providethe protected, cyclized amine. The product was dissolved in a mixture ofTFA and CH₂Cl₂, and allowed to stir at ambient temperature until acomplete reaction was indicated by LCMS. The reaction was concentratedin vacuo and purified by flash chromatography (MeOH/CH₂Cl₂) to providefree amine 5e.

Procedure E: Compounds 5(a-e), the appropriate 6-chloropurine or6-bromopurine, and

DIEA were combined with EtOH or DMF in a small vial and heated to 80° C.The reaction was monitored regularly by LCMS and purified as stated.

Procedure F: A mixture of amide 4b, acetoxyacetyl chloride, and glacialacetic acid was heated to 120° C. and stirred for 2 hours. The cooledreaction was filtered and the solids washed with CH₂Cl₂ to provide thecyclized acetate as a white solid. This material was combined with K₂CO₃in aqueous methanol and stirred for one hour, then concentrated invacuo. The resulting solids were triturated from H₂O to provide 6a as awhite solid.

3-(2-Chlorophenyl)-5-fluoro-2-[(9H-purin-6-ylamino)methyl]-3H-quinazolin-4-one(D-072)

Prepared according to procedure E using 5b (50 mg, 0.165 mmol) and6-chloropurine (26 mg, 0.165 mmol) in 1 mL EtOH. After 5 days, reactionpurified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ). ¹H NMR (DMSO-d₆) δ: 12.99 (br s, 1H); 8.14 (br s, 1H); 8.12 (s,1H); 7.85 (dt, J=5.7, 8.1 Hz., 1H); 7.68-7.79 (m, 3H); 7.57 (t, J=6.2Hz., 1H); 7.57 (d, J=7.7 Hz., 1H); 7.50 (d, J=8.1 Hz., 1H); 7.35 (dd,J=8.4, 10.7 Hz., 1H); 4.15-4.55 (m, 2H). MS (ES): m/z 422 (M+H), 211.

2-[(2-Amino-9H-purin-6-ylamino)methyl]-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one(D-074)

Prepared according to procedure E using 5b (50 mg, 0.165 mmol) and2-amino-6-chloropurine (28 mg, 0.165 mmol) in 1 mL EtOH. After 5 days,reaction purified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min,10-75% acetonitrile/water over 15 min, 100% acetonitrile at 18 min,detector at 220λ). ¹H NMR (DMSO-d₆) δ: 12.13 (br s, 1H); 7.86 (dt,J=5.6, 8.2 Hz., 1H); 7.76-7.83 (m, 2H); 7.68 (br s, 1H); 7.61 (t, J=5.7Hz., 1H); 7.61 (d, J=7.2 Hz., 1H); 7.53 (d, J=8.2 Hz., 1H); 7.35 (dd,J=8.2, 10.9 Hz., 1H); 5.66 (br s, 2H); 4.16-4.50 (m, 1H); 4.09 (q, J=5.3Hz., 2H). MS (ES): m/z 437 (M+H), 219.

5-Methyl-2-[(9H-purin-6-ylamino)methyl]-3-o-tolyl-3H-quinazolin-4-one(D-071)

Prepared according to procedure E using 6-chloropurine (11 mg, 0.072mmol) and 5a (20 mg, 0.072 mmol). After 5 days, the reaction wasquenched with water and the resulting suspension filtered. The solidswere purified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ). ¹H NMR (DMSO-d₆) δ: 12.98 (br s, 1H); 8.14 (br s, 1H); 8.10 (s,1H); 7.58-7.79 (m, 2H); 7.37-7.48 (m, 4H); 7.26-7.36 (m, 2H); 3.93-4.39(m, 2H); 2.75 (s, 3H); 2.18 (s, 3H). MS (ES): m/z 398 (M+H), 199.

2-[(2-Amino-9H-purin-6-ylamino)methyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-073)

Prepared according to procedure E using 5a (189 mg, 0.677 mmol) and2-amino-6-chloropurine (115 mg, 0.677) in 3 mL EtOH. After 3 days, thereaction was filtered to remove excess purine and the filtrate purifiedby HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ) to provide 7 mg of the product as the TFA salt. ¹H NMR (DMSO-d₆)δ: 8.88 (br s, 1H); 8.21 (s, 1H); 7.71 (t, J=7.7 Hz., 1H); 7.45-7.56 (m,2H); 7.38-7.44 (m, 3H); 7.35 (d, J=7.5 Hz., 1H); 7.30 (br s, 1H); 4.40(dd, J=4.5, 17.5 Hz., 1H); 4.27 (dd, J=5.3, 17.4 Hz., 1H); 2.75 (s, 3H);2.09 (s, 3H). MS (ES): m/z 413 (M+H), 207, 163.

2-[(2-Fluoro-9H-purin-6-ylamino)methyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-076)

Prepared according to procedure E using 5a (20 mg, 0.072 mmol) and2-fluoro-6-chloropurine (16 mg, 0.094 mmol) in 1 mL EtOH. After 18hours, the reaction was purified by HPLC (C18 Luna column, 4.6×250 mm,4.7 mL/min, 10-75% acetonitrile/water over 15 min, 100% acetonitrile at18 min, detector at 220λ) and subsequently recrystallized from EtOH toprovide 14 mg of the product as a yellow solid. ¹H NMR (DMSO-d₆) δ:13.12 (br s, 1H); 8.40 (br s, 1H); 8.15 (s, 1H); 7.66 (t, J=7.7 Hz, 1H);7.35-7.49 (m, 4H); 7.31 (d, J=7.2 Hz., 1H); 4.00-4.22 (m, 2H); 3.17 (s,1H); 2.74 (s, 3H); 2.18 (s, 3H). MS (ES): m/z 416 (M+H), 208.

(2-Chlorophenyl)-dimethylamino-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-075)

D-015 (100 mg, 0.228 mmol) was combined with ammonium hydroxide (28-30%,1 mL) in DMF (2 mL) and heated to 80° C. After 2 days, the reaction waspurified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ) to provide the product as a yellow solid, −2 mg. ¹H NMR (DMSO-d₆)δ: 13.52 (br s, 1H); 8.46 (s, 1H); 8.42 (s, 1H); 7.69 (dd, J=2.1, 7.3Hz, 1H); 7.62 (dd, J=1.6, 7.6 Hz., 1H); 7.61 (t, J=8.0 Hz., 1H);7.37-7.48 (m, 2H); 7.05 (d, J=7.9 Hz., 1H); 6.96 (d, J=7.8 Hz., 1H);4.32-4.45 (m, 2H); 2.80 (s, 6H). MS (ES): m/z 464 (M+H), 232.

5-(2-Benzyloxyethoxy)-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-078)

To a solution of 2-benzyloxyethanol (0.3 mL) in DMF (1.0 mL) was addedNaH (50 mg, 2.08 mmol). After stirring for 5 minutes, 0.5 mL was addedto a solution of D-015 (50 mg, 0.114 mmol) in anhydrous DMF (0.75 mL).The reaction was heated to 50° C. and stirred for 3 days. Purificationby HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ) provided the product as a heterogenous solid, 150 μg. MS (ES): m/z571 (M+H), 481.

6-Aminopurine-9-carboxylic acid3-(2-chlorophenyl)-5-fluoro-4-oxo-3,4-dihydro-quinazolin-2-ylmethylester (D-079)

To a solution of 3b (20 mg, 0.066 mmol) in CH₂Cl₂ (500 μL) at 0° C. wasadded phosgene (2M/toluene, 36 μL, 0.072 mmol), followed by adenine (10mg, 0.072 mmol), and DIEA (25 μL, 0.145 mmol). The reaction was allowedto attain ambient temperature and stir for 8 days. Purification by HPLC(C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75% acetonitrile/water over15 min, 100% acetonitrile at 18 min, detector at 220λ) provided theproduct as a mixture. ¹H NMR (DMSO-d₆) δ: 11.04 (br s, 1H); 8.61 (s,1H); 8.40 (s, 1H); 7.85-7.95 (m, 1H); 7.76 (dd, J=5.4, 9.6 Hz, 1H);7.70-7.78 (m, 1H); 7.52-7.63 (m, 3H); 7.38 (dt, J=8.3, 10.6 Hz., 1H);4.76-4.89 (m, 2H). MS (ES): m/z 466 (M+H), 331, 305.

N-[3-(2-Chlorophenyl)-5-fluoro-4-oxo-3,4-dihydroquinazolin-2-ylmethyl]-2-(9H-purin-6-ylsulfanyl)acetamide(D-077)

(9R-Purin-6-ylsulfanyl)-acetic acid (63 mg, 0.296 mmol), 5b (108 mg,0.355 mmol), EDC (68 mg, 0.355 mmol), HOBT (48 mg, 0.355 mmol), DIEA (62μL, 0.355 mmol), and DMF (1 mL) were combined in a flask and stirred atambient temperature for one hour. The reaction was diluted with EtOAc(20 mL) and washed with dilute brine (2×13 mL). The organic phase wasconcentrated in vacuo and chromatographed in 5% MeOH/CH₂Cl₂ to providethe 91 mg of the product as a viscous, peach foam. ¹H NMR (DMSO-d₆) δ:12.88 (br s, 1H); 8.72 (s, 1H); 8.62 (t, J=5.0 Hz, 1H); 8.49 (s, 1H);7.88 (dt, J=5.6, 8.2 Hz, 1H); 7.73-7.78 (m, 1H); 7.67-7.72 (m, 1H);7.57-7.65 (m, 2H); 7.38 (d, J=8.1 Hz., 1H); 7.36 (dd, J=8.3, 11.1 Hz.,1H); 4.11-4.24 (m, 2H); 3.96 (dd, J=5.0, 17.4 Hz, 1H); 3.78 (dd, J=5.2,17.4 Hz, 1H). MS (ES): m/z 496 (M+H), 248.

2-[1-(2-Fluoro-9H-purin-6-ylamino)ethyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-080)

Prepared according to procedure E using 5c (50 mg, 0.17 mmol) and2-fluoro-6-chloropurine (35 mg, 0.204 mmol) in 1.2 mL EtOH. Purificationby HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ) provided two atropisomers as white solids. Data for one of thesefollows: ¹H NMR (DMSO-d₆) δ: 8.48 (br d, J=6.4 Hz, 1H); 8.17 (s, 1H);7.69 (t, J=7.8 Hz, 1H); 7.53 (d, J=7.8 Hz, 1H); 7.44 (d, J=7.8 Hz, 2H);7.33 (d, J=7.2 Hz, 2H); 7.07 (br t, J=7.2 Hz, 1H); 4.80 (br t, J=6.8 Hz,1H); 2.74 (s, 3H); 2.09 (s, 3H); 1.38 (d, J=6.7 Hz, 3H). MS (ES): m/z430 (M+H), 215.

5-Methyl-2-[1-(9H-purin-6-ylamino)ethyl]-3-o-tolyl-3H-quinazolin-4-one(D-081)

Prepared according to procedure E using 5c (50 mg, 0.17 mmol) and6-chloropurine (32 mg, 0.204 mmol) in 1.2 mL EtOH. Purification by HPLC(C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75% acetonitrile/water over15 min, 100% acetonitrile at 18 min, detector at 220λ) provided twoatropisomers as yellow solids. Data for one of these follows: ¹H NMR(DMSO-d₆) δ: 8.39 (br s, 1H); 8.34 (s, 1H); 8.18 (s, 1H); 7.71 (t, J=7.7Hz, 1H); 7.56 (d, J=7.9 Hz, 1H); 7.49 (d, J=6.9 Hz, 1H); 7.28-7.43 (m,3H); 7.20 (br s, 1H); 5.06 (br s, 1H); 2.73 (s, 3H); 2.04 (s, 3H); 1.51(d, J=6.6 Hz, 3H). MS (ES): m/z 412 (M+H), 206.

2-(1-(2-Amino-9H-purin-6-ylamino)ethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-081a)

Synthesized according to procedure E using compound 5c (61 mg, 0.209mmol) and 2-amino-6-chloropurine (43 mg, 0.251 mmol) in 1 mL EtOH.Purification by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at2201) provided a white solid consisting of a mixture of two atropisomers(in brackets in nmr). ¹H NMR (DMSO-d₆) δ: 8.93-9.02 (m, 1H); [8.19 (s),8.15 (s), 1H]; [7.76 (t, J=8.1 Hz), 7.73 (t, J=7.8 Hz), 1H]; [7.64 (d,J=7.9 Hz), 7.57 (d, J=8.0 Hz), 1H]; [7.50 (d, J=7.7 Hz), 7.45 (d, J=7.8Hz), 1H]; 7.29-7.40 (m, 3H); [7.23 (t, J=7.5 Hz), 7.15-7.22 (m), 1H];[7.09 (t, J=7.5 Hz), 6.98 (d, J=7.3 Hz), 1H]; [5.28 (dd, J=7.2, 8.0 Hz),4.96 (pent, J=7.0 Hz), 1H]; 2.75 (s, 3H); [2.10 (s), 1.84 (s), 3H];[1.51 (d, J=6.6 Hz), 1.39 (d, J=6.7 Hz), 3H]. MS (ES): m/z 427 (M+H),214.

5-Methyl-2-(1-(9H-purin-6-ylamino)propyl)-3-o-tolyl-3H-quinazolin-4-one(D-081b)

Synthesized according to procedure E using compound 5d (100 mg, 0.325mmol) and 6-bromopurine (78 mg, 0.390 mmol) in 2 mL EtOH. Purificationby HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at2201) provided two atropisomers as yellow solids. Data for one isomerfollows: ¹H NMR (DMSO-d₆) δ: 8.64 (br s, 1H); 8.44 (s, 1H); 8.27 (s,1H); 7.72 (t, J=7.7 Hz, 1H); 7.56 (d, J=8.0 Hz, 1H); 7.50 (d, J=6.6 Hz,1H); 7.34-7.44 (m, 2H); 7.35 (d, J=7.4 Hz, 1H); 7.18-7.27 (m, 1H);4.85-5.01 (m, 1H); 2.73 (s, 3H); 2.04-2.19 (m, 1H); 1.99 (s, 3H);1.78-1.91 (m, 1H); 0.79 (t, J=7.0 Hz, 3H). MS (ES): m/z 426 (M+H), 213.

2-(1-(2-Fluoro-9H-purin-6-ylamino)propyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-081c)

Synthesized according to procedure E using compound 5d (100 mg, 0.325mmol) and 2-fluoro-6-chloropurine (78 mg, 0.455 mmol) in 2 mL EtOH.Purification by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at2201) provided two atropisomers as off-white solids. Data for oneisomer: ¹H NMR (DMSO-d₆) δ: 8.46 (br d, J=7.1 Hz, 1H); 8.20 (s, 1H);7.71 (t, J=7.7 Hz, 1H); 7.55 (d, J=7.9 Hz, 1H); 7.45 (d, J=7.3 Hz, 1H);7.28-7.37 (m, 3H); 7.00 (t, J=7.3 Hz, 1H); 4.66 (q, J=6.7 Hz, 1H); 2.74(s, 3H); 2.10 (s, 3H); 1.65-1.95 (m, 2H); 0.80 (t, J=7.1 Hz, 3H). MS(ES): m/z 444 (M+H), 222.

2-[1-(2-Amino-9H-purin-6-ylamino)propyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one(Compound 081d)

Synthesized according to procedure E using compound 5d (100 mg, 0.325mmol) and 2-amino-6-bromopurine (104 mg, 0.488 mmol) in 2 mL EtOH.Purification by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at2201) provided an off-white solid consisting of a mixture of twoatropisomers (in brackets in nmr). ¹H NMR (DMSO-d₆) δ: 8.89 (br d, J=7.8Hz, 1H); [8.20 (s), 8.17 (s), 1H]; 7.75 (q, J=7.6 Hz, 1H); [7.62 (d,J=7.9 Hz); 7.57 (d, J=7.8 Hz), 1H]; 7.48 (t, J=7.3 Hz, 1H); 7.25-7.43(m, 4H); 7.15 (br s, 1H); 7.02-7.12 (m, 1H); [5.03-5.15 (m), 4.77-4.87(m), 1H]; 2.74 (s, 3H); [2.11 (s), 1.83 (s), 3H]; 1.65-2.19 (m, 2H);[0.83 (t, J=7.3 Hz), 0.80 (t, J=7.5 Hz), 3H]. MS (ES): m/z 441 (M+H),221.

2-[2-Benzyloxy-1-(9H-purin-6-ylamino)ethyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-081e)

Synthesized according to procedure E using compound 5e (212 mg, 0.413mmol) and 6-bromopurine (107 mg, 0.537 mmol) in 2 mL EtOH. Purificationby HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at2201) provided two atropisomers as brown solids. Data for one isomer: ¹HNMR (DMSO-d₆) δ: 8.45-8.63 (m, 1H); 8.35-8.44 (m, 1H); 8.27 (s, 1H);7.75 (t, J=7.7 Hz, 1H); 7.59 (d, J=7.8 Hz, 1H); 7.30-7.44 (m, 3H);7.21-7.30 (m, 4H); 7.13-7.19 (m, 2H); 6.95-7.07 (m 1H); 5.35-5.45 (m,1H); 5.14-5.26 (m, 1H); 4.43 (s, 2H); 3.94-4.04 (m, 1H); 3.67 (dd,J=6.0, 9.4 Hz, 1H); 2.74 (s, 3H); 2.01 (s, 3H). MS (ES): m/z 518 (M+H),410.

The following compounds of the present invention (D-082 through D-109)were prepared as outlined in Procedure C, using2-chloromethyl-5-methyl-3-o-tolyl-3H-quinazolin-4-one (10 mg), theappropriate nucleophile XH (20 mg, excess), and potassium carbonate (10mg) in DMF (0.25 mL). The reaction mixture was stirred 16 h at roomtemperature, quenched with water, and the crude solid product wascollected by filtration and air dried. The crude material was dissolvedin 0.5 mL of DMSO and purified by reversed-phase HPLC (C18 Luna column,4.6×250 mm, 4.7 mL/min, 10-75% acetonitrile/water over 15 min, 100%acetonitrile at 18 min, detector at 220A). Appropriate fractions wereconcentrated in vacuo to yield the final products.

2-(6-Dimethylaminopurin-9-ylmethyl)-5-methyl-3-O—tolyl-3H-quinazolin-4-one (D-082)

Yield: 8.1 mg. ¹H NMR (300 MHz, d₆-DMSO) δ: 8.13 (s, 1H), 8.11 (s, 1H),7.60 (t, J=7.8 Hz, 1H), 7.54-7.38 (m, 4H), 7.30 (d, J=7.4 Hz, 1H), 7.20(d, J=8.1 Hz, 1H), 5.11 (d, J=17.4 Hz, 1H), 4.76 (d, J=17.4 Hz, 1H),3.33 (s, 6H), 2.73 (s, 3H), 2.20 (s, 3H). LRMS (ES pos.) m/z=426 (M+1).

5-Methyl-2-(2-methyl-6-oxo-1,6-dihydro-purin-7-ylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-083)

Yield: 3.3 mg. ¹H NMR (300 MHz, d₆-DMSO) δ: 12.06 (s, 1H), 8.12 (s, 1H),7.60 (t, J=7.8 Hz, 1H), 7.55-7.38 (m, 4H), 7.30 (d, J=7.4 Hz, 1H), 7.15(d, J=7.9 Hz, 1H), 5.26 (d, J=17.4 Hz, 1H), 4.94 (d, J=17.4 Hz, 1H),2.73 (s, 3H), 2.32 (s, 3H), 2.24 (s, 3H). Alkylation at purine N₇assigned arbitrarily based on downfield shift of methylene protons dueto the carbonyl group. LRMS (ES pos.) m/z=413 (M+1).

5-Methyl-2-(2-methyl-6-oxo-1,6-dihydro-purin-9-ylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-084)

Purified from same reaction mixture as D-083. Yield: 3.6 mg. ¹H NMR (300MHz, d₆-DMSO) 12.17 (s, 1H), 7.96 (s, 1H), 7.63 (t, J=7.8 Hz, 1H),7.57-7.39 (m, 4H), 7.32 (d, J=7.4 Hz, 1H), 7.26 (d, J=8.1 Hz, 1H), 5.08(d, J=17.2 Hz, 1H), 4.70 (d, J=17.2 Hz, 1H), 2.73 (5, 3H), 2.27 (s, 3H),2.17 (s, 3H). LRMS (ES pos.) m/z=413 (M+1).

2-(Amino-dimethylaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-085)

Yield: 6.7 mg. ¹H NMR (300 MHz, d₆-DMSO) δ: 7.66 (s, 1HO, 7.61 (d, J=7.8Hz, 1H), 7.55-7.40 (m, 4H), 7.32-7.26 (m, 2H), 6.74 (s, 2H), 4.94 (d,J=17.2 Hz, 1H), 4.63 (d, J=17.2 Hz, 1H), 4.63 (d, J=17.2 Hz, 1H), 2.97(s, 6H), 2.73 (s, 3H), 2.17 (s, 3H), 2.08 (s, 3H). LRMS (ES pos.)m/z=441 (M+1).

2-(2-Amino-9H-purin-6-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-086)

Yield: 9.5 mg. ¹H NMR (300 MHz, d₆-DMSO) δ: 12.54 (s, 1H), 7.89 (s, 1H),7.69 (t, J=7.8 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H),7.43 (t, J=3.9 Hz, 1H), 7.34=7.26 (m, 4H), 6.16 (s, 2H), 4.32 (ABquartet, J_(AB)=14.8 Hz, Δn=23.7), 2.74 (s, 3H), 2.09 (s, 3H). LRMS (ESpos.) m/z=430 (M+1).

2-(4-Amino-1,3,5-triazin-2-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-087)

Yield: 5.8 mg. ¹H NMR (300 MHz, d₆-DMSO) δ: 8.10 (s, 1H), 7.70 (t, J=7.8Hz, 1H), 7.58 (s, 1H), 7.52 (d, J=8.0 Hz, 1H), 7.48-7.26 (m, 6H), 4.08(s, 2H), 2.73 (s, 3H), 2.09 (s, 3H). LRMS (ES pos.) m/z=391 (M+1).

5-Methyl-2-(7-methyl-7H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-088)

Yield: 3.1 mg. ¹H NMR (300 MHz, d₆-DMSO) δ: 8.52 (s, 1H), 8.49 (s, 1H),7.70 (t, J=7.8 Hz, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.45 (d, J=7.1 Hz, 1H),7.35-7.20 (m, 4H), 4.41 (AB quartet, J_(AB)=15.3 Hz, Δν=19.2 Hz), 4.08(s, 3H), 2.73 (s, 3H), 2.12 (s, 3H). LRMS (ES pos.) m/z=406 (M+1).

5-Methyl-2-(2-oxo-1,2-dihydro-pyrimidin-4-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-089)

Yield: 2.4 mg. ¹H NMR (300 MHz, d₆-DMSO) δ: 11.49 (s, 1H), 7.70 (t,J=7.8 Hz, 1H), 7.60 (brt, J=6.0 Hz, 1H), 7.53-7.48 (m, 2H), 7.46-7.28(m, 4H), 6.31 (d, J=6.7 Hz, 1H), 4.05 (s, 2H), 2.73 (s, 3H), 2.12 (s,3H). LRMS (ES pos.) m/z=391 (M+1).

5-Methyl-2-purin-7-ylmethyl-3-o-tolyl-3H-quinazolin-4-one (D-090)

¹H NMR (300 MHz, d₆-DMSO) δ: 9.04 (s, 1H), 8.97 (s, 1H), 8.48 (s, 1H),7.65-7.54 (m, 2H), 7.53-7.39 (m, 3H), 7.31 (d, J=7.4 Hz, 1H), 7.13 (d,J=8.0 Hz, 1H), 5.31 (d, J=17.6 Hz, 1H), 5.16 (d, J=17.6 Hz, 1H), 2.73(s, 3H), 2.09 (s, 3H). Alkylation at purine N7 was determined by NOEenhancement between the purine 6-position proton and methylene protonson the linker between the purine and quinazolinone groups. LRMS (ESpos.) m/z=383 (M+1).

5-Methyl-2-purin-9-ylmethyl-3-o-tolyl-3H-quinazolin-4-one (D-091)

From same reaction that produced D-090. ¹H NMR (300 MHz, d₆-DMSO) δ:9.17 (s, 1H), 8.86 (s, 1H), 8.55 (s, 1H), 7.59 (t, J=7.8 Hz, 1H),7.55-7.42 (m, 4H), 7.30 (d, J=7.4 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 5.26(d, J=17.5 Hz, 1H), 4.92 (d, J=17.5 Hz, 1H), 2.73 (s, 3H), 2.19 (s, 3H).Alkylation at purine N9 suggested by the lack of NOE enhancement betweenpurine 6-position protons and the linker methylene protons. LRMS (ESpos.) m/z=383 (M+1).

5-Methyl-2-(9-methyl-9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-092)

¹H NMR (300 MHz, d₆-DMSO) δ: 8.52 (s, 1H), 8.42 (s, 1H), 7.69 (t, J=7.7Hz, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.44 (d, J=7.6 Hz, 1H), 7.36-7.27 (m,4H), 4.38 (AB quartet, J_(AB)=15.5 Hz, Δν=21.0 Hz), 3.80 (s, 3H), 2.73(s, 3H), 2.12 (s, 3H). LRMS (ES pos.) m/z=429 (M+1).

2-(2,6-Diamino-pyrimidin-4-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-093)

¹H NMR (300 MHz, d₆-DMSO) δ: 7.70 (t, J=7.7 Hz, 1H), 7.54 (d, J=8.0 Hz,1H), 7.45-7.27 (m, 5H), 6.22 (br s, 1H), 5.80 (br s, 1H), 3.99 (ABquartet, J_(AB)=14.6 Hz, Δν=26.9 Hz, 2H), 2.73 (s, 3H), 2.08 (s, 3H).LRMS (ES pos.) m/z=405 (M+1).

5-Methyl-2-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-094)

¹H NMR (300 MHz, d₆-DMSO) δ: 8.57 (s, 1H), 7.73 (t, J=7.8 Hz, 1H),7.55-7.35 (m, 4H), 7.18 (s, 1H), 4.27 (s, 2H), 2.74 (s, 3H0, 2.55 (s,3H), 2.08 (s, 3H). LRMS (ES pos.) m/z=429 (M+1).

5-Methyl-2-(2-methylsulfanyl-9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-095)

¹H NMR (300 MHz, d₆-DMSO) δ: 13.30 (s, 1H), 8.29 (s, 1H), 7.72 (t, J=7.8Hz, 1H), 7.54 (d, J=7.8 Hz, 1H), 7.47 9d, J=6.3 Hz, 1H), 7.38-7.26 (m,4H), 4.34 (AB quartet, J_(AB)=16.1 Hz, Δν=23.6 Hz, 2H), 2.74 (s, 3H),2.32 (s, 3H), 2.10 (s, 3H). LRMS (ES pos.) m/z=461 (M+1).

2-(2-Hydroxy-9H-purin-6-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-096)

¹H NMR (300 MHz, d₆-DMSO) δ: 8.08 (s, 1H), 7.69 (t, J=7.8 Hz, 1H), 7.50(brd, J=t.8 Hz, 2H), 7.33-7.50 (m, 4H), 4.28 (AB quartet, J_(AB)=15.5Hz, Δν=21.3 Hz, 2H), 2.74 (s, 3H), 2.12 (s, 3H). LRMS (ES pos.) m/z=431(M+1).

5-Methyl-2-(1-methyl-1H-imidazol-2-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-097)

¹H NMR (300 MHz, d₆-DMSO) δ: 7.69 t, J=7.8 Hz, 1H), 7.46-7.37 (m, 5H),7.32 (d, J=7.3 Hz, 1H), 7.20 (d, J=1.0 Hz, 1H), 6.48 (d, J=1.0 Hz), 3.83(AB quartet, J_(AB)=15.0 Hz, Δν=18.8 Hz, 1H), 3.55 (s, 3H), 2.73 (s,3H), 2.09 (s, 3H). LRMS (ES pos.) m/z=364 (M+1).

5-Methyl-3-o-tolyl-2-(1H-[1,2,4]triazol-3-ylsulfanylmethyl)-1H-quinazolin-4-one(D-098)

¹H NMR (300 MHz, d₆-DMSO) δ: 13.98 (s, 1H), 8.47 (s, 1H), 7.70 (t, J=7.8Hz, 1H), 7.49 (d, J=7.9 Hz, 1H), 7.44-7.31 (m, 5H), 4.04 (AB quartet,J_(AB)=15.5 Hz, Δν=19.1 Hz, 1H), 2.74 (s, 3H), 2.10 (s, 3H). LRMS (ESpos.) m/z=364 (M+1).

2-(2-Amino-6-chloro-purin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-099)

LRMS (ES pos.) 432 (M+1).

2-(6-Aminopurin-7-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-100)

¹H NMR (300 MHz, d₆-DMSO) δ: 8.19 (s, 3H), 7.66 (t, J=7.8 Hz, 1H),7.59-7.43 (m, 5H), 7.34 9d, J=7.4 Hz, 1H), 7.23 (d, J=8.0 Hz, 1H), 6.90(s, 2H), 5.21 (AB quartet, J_(A8)=17.4 Hz, Δν=22.1 Hz, 2H), 2.72 (s,3H), 1.93 (s, 3H). Alkylation at purine N7 was confirmed by NOEenhancements between the following protons: 1) Exocyclic amine andmethylene protons; 2) Exocyclic amine and toluoyl methyl protons. LRMS(ES pos.) m/z=398 (M+1).

2-(7-Amino-1,2,3-triazolo[4,5-d]pyrimidin-3-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-101)

¹H NMR (300 MHz, d₆-DMSO) δ: 8.43 (br s, 1H), 8.19 (s, 1H), 8.10 (br s,1H), 7.62 (t, J=7.8 Hz, 1H), 7.49-7.28 (m, 5H), 7.22 (d, J=8.1 Hz, 1H),5.49 (d, J=17.0 Hz, 1H), 5.19 (d, J=17.0 Hz, 1H), 2.73 (s, 3H), 2.11 (s,3H). Alkylation at purine N7 determined by similarity to nmr spectrum ofD-030. LRMS (ES pos.) m/z=399 (M+1).

2-(7-Amino-1,2,3-triazolo[4,5-d]pyrimidin-1-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-102)

From same reaction mixture as D-101. ¹H NMR (300 MHz, d₆-DMSO) δ: 8.27(s, 1H), 8.20 (br s, 1H), 8.05 (br s. 1H), 7.70 (t, J=7.8 Hz, 1H),7.47-7.26 (m, 6H), 5.61 (AB quartet, J_(AB)=16.0 Hz, Δν=20.7 Hz, 2H),2.75 (s, 3H), 1.98 (s, 3H)). Alkylation at purine N7 determined bysimilarity to nmr spectrum of D-100. LRMS (ES pos.) m/z=399 (M+1).

2-(6-Amino-9H-purin-2-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-103)

¹H NMR (300 MHz, d₆-DMSO) δ: 12.62 (s, 1H), 7.93 (s, 1H), 7.69 (t, J=7.7Hz, 1H), 7.51 (d, J=8.1 Hz, 1H), 7.42 (dd, J=7.6, 1.7 Hz, 1H), 7.35-7.15(m, 6H), 4.12 (AB quartet, J_(AB)=14.5 Hz, Δν=18.2 Hz, 2H), 2.73 (s,3H), 2.10 (s, 3H). LRMS (ES pos.) m/z=430 (M+1).

2-(2-Amino-6-ethylamino-pyrimidin-4-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-104)

¹H NMR (300 MHz, d₆-DMSO) δ: 7.70 (T, J=7.8 Hz, 1H), 7.53 (d, J=8.0 Hz,1H), 7.44-7.31 (m, 5H), 6.69 (br s, 1H), 5.83, (br s, 2H), 5.61 (s, 1H),4.03 (d, J=14.6 Hz, 1H), 3.95 (d, J=14.6 Hz, 1H), 3.22-3.11 (m, 2H),2.73 (s, 3H), 2.08 (s, 3H), 1.06 (t, J=7.1 Hz, 3H). LRMS (ES pos.)m/z=433 (M+1).

2-(3-Amino-5-methylsulfanyl-1,2,4-triazol-1-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-105)

Yield: 5.0 mg. ¹H NMR (300 MHz, d₄-MeOH) δ: 7.67 (t, J=7.8 Hz, 1H),7.55-7.37 (m, 4H), 7.35-7.27 (m, 2H), 4.77 (d, J=17.1 Hz, 1H), 4.60 (d,J=17.1 Hz, 1H), 2.80 (s, 3H), 2.43 (s, 3H), 2.14 (s, 3H). LRMS (ES pos.)m/z=393 (M+1).

2-(5-Amino-3-methylsulfanyl-1,2,4-triazol-1-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one(D-106)

Yield: 0.6 mg. Purified from same reaction mixture as D-105. ¹H NMR (300MHz, d₄-MeOH) δ: 7.67 (t, J=7.8 Hz, 1H), 7.50-7.24 (m, 6H), 4.83 (d,J=16.5 Hz, 1H), 4.70 (d, J=16.5 Hz, 1H), 2.79 (s, 3H), 2.47 (s, 3H),2.14 (s, 3H). LRMS (ES pos.) m/z=393 (M+1).

5-Methyl-2-(6-methylaminopurin-9-ylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-107)

Yield: 5.0 mg ¹H NMR (300 MHz, d₄-MeOH) δ: 8.17 (s, 1H), 8.03 (s, 1H),7.54-7.43 (m 4H), 7.31-7.23 (m, 2H), 5.14 (d, J=17.5 Hz, 1H), 4.90 (d,J=17.5 Hz, 1H), 3.14 (br s, 3H), 2.79 (s, 3H), 2.22 (s, 3H). LRMS (ESpos.) m/z=412 (M+1).

2-(6-Benzylaminopurin-9-ylmethyl)-5-methyl-3-O—tolyl-3H-quinazolin-4-one (D-108)

Yield: 6.7 mg. ¹H NMR (300 MHz, d₄-MeOH) δ: 8.13 (s, 1H), 8.04 (s, 1H),7.58 (t, J=7.8 Hz, 1H), 7.51-7.21 (m, 11H), 5.15 (d, J=17.5 Hz, 1H),4.91 (d, J=17.5 Hz, 1H), 4.83 (s, 2H, under H₂O Peak), 2.79 (s, 3H),2.22 (s, 3H). LRMS (ES pos.) m/z=488 (M+1).

2-(2,6-Diaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-1H-quinazolin-4-one(D-109)

Doubled the amounts of all reactants. Yield: 14 mg. ¹H NMR (300 MHz,d₆-DMSO) δ: 8.53 (br s, 2H), 8.01 (s, 1H), 7.64 (t, J=7.8 Hz, 1H),7.53-7.40 (m, 4H), 7.33 (d, J=7.4 Hz, 1H), 7.27 9d, J=7.9 Hz, 1H), 4.96(d, J=17.5 Hz, 1H), 4.64 (d, J=17.5 Hz, 1H), 2.74 (s, 3H), 2.17 (s, 3H).LRMS (ES pos.) m/z=413 (M+1).

Compounds D-110 through D-115 of the following general structure wereprepared from the following Intermediates E-1 through E-3.

Intermediate E-15-Methyl-2-(9H-purin-6-ylsulfanylmethyl)-3,1-benzoxazin-4-one

Step 1. A suspension of 6-methylanthranilic acid (2 g, 13.2 mmol) inchloroacetyl chloride (12 mL, large excess) was stirred at 115° C. in asealed vial for 30 min. The resulting solution was cooled to roomtemperature and treated with ether (˜5 mL). After cooling at 4° C.overnight, the resulting tan precipitate was collected by filtration,washed with ether, and dried in vacuo to yield the chloro intermediate(1.39 g, 50%). ¹H NMR (300 MHz, CDCl₃) δ: 7.67 (t, J=7.8 Hz, 1H), 7.46(d, J=7.9 Hz, 1H), 7.35 (d, J=7.6 Hz, 1H), 4.39 (s, 2H), 2.81 (s, 3H).LRMS (ES pos.) m/z=210, (M+1).

Step 2. A mixture of the chloro intermediate (50 mg, 0.25 mmol),6-mercaptopurine monohydrate (43 mg, 0.25 mmol), and potassium carbonate(25 mg, 0.25 mmol) in dry DMF (0.5 mL) was stirred at room temperaturefor 30 min. The mixture was poured into ethyl acetate (20 mL) and allinsoluble material was filtered off and discarded. The filtrate wasconcentrated in vacuo to remove all ethyl acetate, and the residue wastreated with ether, resulting in a light orange precipitate. Theprecipitate was collected by filtration, washed with ether, and dried invacuo to afford Intermediate E-1 (41 mg, 51%). ¹H NMR (300 MHz, d₆-DMSO)δ: 8.64 (s, 1H), 8.39 (s, 1H), 7.73 (t, J=7.8 Hz, 1H), 7.44-7.37 (m,2H), 4.69 (s, 2H), 2.69 (s, 3H). LRMS (ES pos.) m/z=326 (M+1).

Intermediate E-2

A solution of 2-nitroacetanilide (1.0 g, 5.6 mmol) in EtOH was purgedwith nitrogen, treated with Pd(OH)₂ (20% by wt. on C, 200 mg, cat.), andshaken for 2 h under H₂ (20 psi). The catalyst was removed by filtrationthrough a 0.22 um cellulose acetate membrane (Corning), and the filtratewas concentrated in vacuo to afford the white crystalline solid product(800 mg, 96%). ¹H NMR (300 MHz, d₆-DMSO) δ: 9.12 (s, 1H), 7.14 (dd,J=7.8, 1.3 Hz, 1H), 6.88 (dt, J=7.6, 1.5 Hz, 1H), 6.70 (dd, J=8.0, 1.3Hz, 1H), 6.52 (dt, J=7.5, 1.4 Hz, 1H), 4.85 (br s, 2H), 2.03 (s, 3H).LRMS (ES pos.) m/z=151 (M+1).

Intermediate E-3

A mixture of 2-fluoro-nitrobenzene (1.41 g, 10 mmol) and NaHCO₃ in EtOH(20 mL) was treated with (N,N,N′-trimethyl)-1,2-diaminoethane (1.1 g, 11mmol) and was stirred 16 h at 80° C. Solvent was removed in vacuo,residue was treated with 0.1 M NaOH (120 mL), and the mixture wasextracted with ethyl acetate (2×50 mL). The organic layers were combinedand washed with 20 mL of water (1×) and brine (2×), dried with sodiumsulfate, and concentrated in vacuo to an orange liquid (2.2 g, 100%;ESMS: m/z=224, M+1).

This intermediate was dissolved in EtOH, the solution was purged withnitrogen, treated with Pd(OH)₂ (20% by wt. on C, 180 mg, cat.), andshaken for 2 h under H₂ (50 psi). The catalyst was removed by filtrationthrough a 0.22 um cellulose acetate membrane (Corning), and the filtratewas concentrated in vacuo to afford the red liquid product E-3 (1.8 g,95%). ¹H NMR (300 MHz, CDCl₃) δ: 8.64 (s, 1H), 7.03 (dd, J=8.3, 1.4 Hz,1H), 6.91 (ddd, J=7.6, 7.2, 1.4 Hz, 1H), 6.73-6.67 (m, 2H), 4.20 (br s,2H), 2.95 (t, J=6.7 Hz, 2H), 2.68 (s, 3H), 2.41 (t, J=6.7 Hz, 1H), 2.26(s, 6H). LRMS (ES pos.) m/z=194 (M+1).

Compounds D-110 through D-115 were prepared as follows:

5-Methyl-2-(9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one(D-110)

A mixture of Intermediate E-1 (40 mg) and o-toluidine (0.3 mL, largeexcess) was warmed at 100° C. in a sealed vial for 16 h. The reactionmixture was cooled, treated with 1N HCl (2 mL) and ether (2 mL), and theresulting gray precipitate was collected by filtration, washed withether, and air dried (19 mg crude). The crude solid was dissolved in 0.5mL DMSO and purified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min,10-75% acetonitrile/water over 15 min, 100% acetonitrile at 18 min,detector at 220λ). Appropriate fractions were concentrated in vacuo toyield the final product as a white solid (4 mg). ¹H NMR (300 MHz,d₆-DMSO) δ: 13.52 (s, 1H), 8.47 (s, 1H), 8.43 (s, 1H), 7.69 (t, J=7.8Hz, 1H), 7.50 (d, J=7.9 Hz, 1H), 7.46-7/43 (m, 1H), 7.37-7.25 (m, 4H),4.37 (AB quartet, J=15.4 Hz, Δν=22.4 Hz, 2H), 2.74 (5, 3H), 2.12 (5,3H). LRMS (ES pos.) m/z=415 (M+1).

3-Isobutyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-111)

A mixture of Intermediate E-1 (40 mg) and isobutylamine (0.4 mL, largeexcess) was warmed at 120° C. in a sealed vial for 16 h. Excessisobutylamine was allowed to evaporate, residue was dissolved in 1 mLDMSO and purified in two portions by HPLC (C18 Luna column, 4.6×250 mm,4.7 mL/min, 10-75% acetonitrile/water over 15 min, 100% acetonitrile at18 min, detector at 220λ). Appropriate fractions were concentrated invacuo to yield the final product as a white solid (4 mg). ¹H NMR (300MHz, d₆-DMSO) δ: 13.75 (br s, 1H), 8.73 (s, 1H), 8.50 (s, 1H), 7.63 (t,J=7.7 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.28 (d, J=7.3 Hz, 1H), 4.96 (s,2H), 4.00 (d, J=7.5 Hz, 2H), 2.77 (s, 3H), 2.30-2.15 (m, 1H), 0.98 (d,J=6.7 Hz, 1H). LRMS (ES pos.) m/z=381 (M+1).

N-{2-[5-Methyl-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]-phenyl}-acetamide(D-112)

A mixture of Intermediate E-1 (80 mg, 0.25 mmol) and Intermediate E-2(75 mg, 0.5 mmol, 2 eq) was warmed until melted in a sealed vial using aheat gun. The reaction mixture was triturated with ether and the solidswere collected by filtration. The crude material was dissolved in 1 mLDMSO and purified in two portions by HPLC (C18 Luna column, 4.6×250 mm,4.7 mL/min, 10-75% acetonitrile/water over 15 min, 100% acetonitrile at18 min, detector at 220λ). Appropriate fractions were concentrated invacuo to yield the final product as a white solid. ¹H NMR (300 MHz,d₆-DMSO) δ: 13.52 (s, 1H), 9.52 (s, 1H), 8.48 (s, 3H), 8.42 (s, 3H),8.02 (d, J=8.0 Hz, 1H), 7.69 (t, J=7.8 Hz, 1H), 7.51 (d, J=7.9 Hz, 1H),7.45-7.37 (m, 2H), 7.31 (d, J=7.3 Hz, 1H), 7.19 (t, J=7.5 Hz, 1H), 4.38(s, 2H), 2.74 (s, 3H), 1.93 (s, 3H). LRMS (ES pos.) m/z=458 (M+1).

5-Methyl-3-(E-2-methyl-cyclohexyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-113)

A mixture of Intermediate E-1 (80 mg, 0.25 mmol) andtrans-2-methyl-1-aminocyclohexane (0.25 mL, large excess) was warmed ina sealed vial at 100° C. for 16 h. The reaction mixture was trituratedwith ether and the solids were collected by filtration. The crudematerial was dissolved in 0.5 mL DMSO and purified by HPLC(C18 Lunacolumn, 4.6×250 mm, 4.7 mL/min, 10-75% acetonitrile/water over 15 min,100% acetonitrile at 18 min, detector at 220λ). Appropriate fractionswere concentrated in vacuo to yield the final product as a white solid(1.5 mg). ¹H NMR (300 MHz, d₆-DMSO) δ: 13.5 (br s, 1H), 8.82 (s, 1H),8.51 (s, 1H), 7.63 (t, J=7.7 Hz, 1H), 7.43 (d, J=7.9 Hz, 1H), 7.27 (d,J=7.4 Hz, 1H), 5.11 (d, J=14.5 Hz, 1H), 3.78-3.69 (m, 1H), 2.73 (s, 3H),2.55-2.40 (m, 3H), 1.88-1.46 (m, 4H), 1.31-1.11 (m, 1H), 0.90-0.65 (m,1H), 0.74 (d, J=6.7 Hz, 3H). LRMS (ES pos.) m/z=421 (M+1).

2-[5-Methyl-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]-benzoicacid (D-114)

A mixture of Intermediate E-1 (80 mg, 0.25 mmol) methyl anthranilate(0.25 mL, large excess) was warmed in a sealed vial at 100° C. for 16 h.The reaction mixture was triturated with ether and the solids werecollected by filtration. The crude material was dissolved in 0.5 mL DMSOand purified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ). Appropriate fractions were concentrated in vacuo to yield thefinal product as a white solid (8 mg). ¹H NMR (300 MHz, d₆-DMSO) δ:13.51 (s, 1H), 8.51 (s, 1H), 8.42 (s, 1H), 8.11 (dd, J=7.4, 1.1 Hz, 1H),7.88 (dt, J=7.7, 1.4 Hz, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.57 (t, J=7.2 Hz,1H), 7.49-7.35 (m, 3H), 4.58 (d, J=15.5 Hz, 1H), 4.35 (d, J=15.5 Hz,1H), 2.44 (s, 3H). LRMS (ES pos.) m/z=445 (M+1).

3-{2-[(2-Dimethylamino-ethyl)-methyl-amino]-phenyl}-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-115)

A mixture of Intermediate E-1 (40 mg, 0.25 mmol) Intermediate E-3 (0.2mL, large excess) was warmed in a sealed vial at 100° C. for 16 h. Thereaction mixture was triturated with ether and the solids were collectedby filtration. The crude material was dissolved in 1 mL DMSO andpurified by HPLC in two portions (C18 Luna column, 4.6×250 mm, 4.7mL/min, 10-75% acetonitrile/water over 15 min, 100% acetonitrile at 18min, 0.05% TFA in all solvents, detector at 220λ). Appropriate fractionswere concentrated in vacuo to yield the final product as the TFA salt(11 mg). ¹H NMR (300 MHz, d₆-DMSO) δ: 13.4 (br s, 1H), 9.27 (s, 1H),8.52 (s, 1H), 8.44 (s, 1H), 7.72 (t, J=7.8 Hz, 1H), 7.53 (d, J=7.9 Hz,1H), 7.40-7.33 (m, 4H), 7.10-7.04 (m, 1H), 4.42 (s, 3H), 3.5 (m, 2H),3.23-3.03 (m, 3H), 2.75 (s, 3H), 2.68-2.56 (m, 8H). LRMS (ES pos.)m/z=501 (M+1).

Compounds D-116 through D-118 were prepared as follows:

3-(2-Chlorophenyl)-5-methoxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-116)

(R=Me, X═O)

A mixture of D-015 (25 mg) in 0.5 M NaOMe (2 mL in MeOH; large excess)was stirred at 50° C. for 16 h in a sealed vial. The reaction mixturewas cooled to room temperature, treated with water (5 mL), and theresulting precipitate was collected by filtration, washed with water,and air dried. The crude material was dissolved in 0.5 mL DMSO andpurified by HPLC (C18 Luna column, 4.6×250 mm, 4.6×250 mm, 4.7 mL/min,10-75% acetonitrile/water over 15 min, 100% acetonitrile at 18 min,detector at 220λ). Appropriate fractions were concentrated in vacuo toyield the final product as a white solid (5.3 mg). ¹H NMR (300 MHz,d₆-DMSO) δ: 13.52 (s, 1H), 8.48 (s, 1H), 8.44 (br s, 1H), 7.77 (t, J=8.2Hz, 1H), 7.71-7.60 (m, 2H), 7.51-7.34 (m, 2H), 7.23 (d, J=8.2 Hz, 1H),7.10 (d, J=8.4 Hz, 1H), 4.39 (AB quartet, J_(AB)=5.2 Hz, Δν=23.2 Hz,2H), 3.85 (s, 3H). LRMS (ES positive) m/z=451 (M+1).

3-(2-Chlorophenyl)-5-(2-morpholin-4-yl-ethylamino)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-117)

A mixture of D-015 (25 mg) and 4-(aminoeth-2-yl)morpholine (650 mg,large excess) was stirred at 50° C. for 16 h. The crude reaction mixturewas purified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min, 10-75%acetonitrile/water over 15 min, 100% acetonitrile at 18 min, detector at220λ). Appropriate fractions were concentrated in vacuo to yield thefinal product. ¹H NMR (300 MHz, d_(c)-acetone) δ: 8.57 (br s, 1H), 8.47(s, 1H), 8.37 (s, 1H), 7.72 (dd, J=7.7, 1.6 Hz, 1H), 7.65 (dd, J=8.0,1.2 Hz, 1H), 7.57 (t, J=8.1 Hz, 1H), 7.49 (dt, J=7.7, 1.6 Hz, 1H), 7.40(dt, J=7.7, 1.5 Hz, 1H), 6.86 (d, J=7.4 Hz, 1H), 6.82 (d, J=8.3 Hz, 1H),4.55 (d, J=15.0 Hz, 1H), 4.42 (d, J=15.1 Hz, 1H), 4.05-3.90 (m, 4H),3.90 (t, J=6.9 Hz, 2H), 3.75-3.4 (m, 4H), 3.54 (t, J=6.9 Hz, 2H). LRMS(ES positive) m/z=549 (M+1).

3-Benzyl-5-methoxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one(D-118)

A mixture of D-043 (25 mg) in 0.5 M NaOMe (2 mL in MeOH; large excess)was stirred at 50° C. for 16 h in a sealed vial. The reaction mixturewas treated with 1 N HCl (1 mL) and aliquots of this solution (0.5 mLeach) were purified by HPLC (C18 Luna column, 4.6×250 mm, 4.7 mL/min,10-75% acetonitrile/water over 15 min, 100% acetonitrile at 18 min,detector at 220λ). Appropriate fractions were concentrated in vacuo toyield the final product as a white solid (6.6 mg). ¹H NMR (300 MHz,d₆-DMSO) δ: 13.57 (s, 1H), 8.60 (S, 1H), 8.45 (s, 1H), 7.72 (t, J=8.1Hz, 1H), 7.42-7.30 (m, 2H), 7.30-7.19 (m, 3H), 7.15 (d, J=8.0 Hz, 1H),7.06 (d, J=8.3 Hz, 1H), 5.43 (s, 2H), 4.80 (s, 2H), 3.87 (s, 3H). LRMS(ES positive) m/z=431 (M+1).

Compound D-999 (Comparative)3-(2-Chlorophenyl)-2-(1H-pyrazolo[3,4-d]pyrimidin-4-ylsulfanylmethyl)-3H-quinazolin-4-one

An analog compound,3-(2-chlorophenyl)-2-(1H-pyrazolo[3,4-d]pyrimidin-4-ylsulfanylmethyl)-3H-quinazolin-4-one,also was synthesized generally in accordance with the described methods,except that a 4-mercapto-1H-pyrazolo[3,4-d]pyrimidine was substitutedfor the mercaptopurine in the final step.

EXAMPLE 11

Biochemical Assays of PI3K Potency and Selectivity

A. Biochemical Assay Using 20 μM ATP

Using the method described in Example 2, above, compounds of theinvention were tested for inhibitory activity and potency against PI3Kδ,and for selectivity for PI3Kδ versus other Class I PI3K isozymes. InTable 2, IC₅₀ values (μM) are given for PI3Kα (“Alpha”), PI3Kβ (“Beta”),PI3γ (“Gamma”), and PI3Kδ (“Delta”). To illustrate selectivity of thecompounds, the ratios of the IC₅₀ values of the compounds for PI3Kα,PI3β, and PI3Kγrelative to PI3Kδ are given, respectively, as“Alpha/Delta Ratio,” “Beta/Delta Ratio,” and “Gamma/Delta Ratio.”

The initial selectivity assays were done identically to the selectivityassay protocol in Example 2, except using 100 μL Ecoscint for radiolabeldetection. Subsequent selectivity assays were done similarly using thesame 3× substrate stocks except they contained 0.05 mCi/mL γ[³²P]ATP and3 mM PIP₂. Subsequent selectivity assays also used the same 3× enzymestocks, except they now contained 3 nM of any given PI3K isoform.

For all selectivity assays, the test compounds were weighed out anddissolved into 10-50 mM stocks in 100% DMSO (depending on theirrespective solubilities) and stored at −20° C. Compounds were thawed (toroom temperature or 37° C.), diluted to 300 μM in water from which a3-fold dilution series into water was done. From these dilutions, 20 μLwas added into the assay wells alongside water blanks used for theenzyme (positive) control and the no enzyme (background) control. Therest of the assay was essentially done according to the selectivityassay protocol in Example 2.

For those cases in which the greatest concentration used in the assay,i.e., 100 μM, did not inhibit activity of the enzyme by at least 50%,the table recites the percent activity remaining at that concentration(i.e., at 100 μM). In these cases, the true activity ratio(s) for thecompounds cannot be calculated, since one of the required IC₅₀ values ismissing. However, to provide some insight into the characteristics ofthese compounds, a hypothetical activity ratio is calculated using 100μM substituted for the missing value. In such cases, the selectivityratio must in fact be greater than the hypothetical value, and this isindicated by use of a greater than (>) symbol.

TABLE 2 Alpha/Delta Beta/Delta Gamma/Delta Compound Alpha IC₅₀ Beta IC₅₀Delta IC₅₀ Gamma IC₅₀ Ratio Ratio Ratio D-000 86% 74% 0.33 7.7 >302 >30223 D-001 83% 45 68 >1.5 0.66 D-002 88% 78% 44 >2.3 >2.3 D-003 92 53% 422 >24 D-004 93% 89% 64 >2 >1.6 D-005 89% 46 0.8 >121 56 D-006 78% 60.15 >652 38 D-007 82% 30 0.16 >619 188 D-008 82% 68 1.2 >85 57 D-009 826 0.12 683 50 D-010 48 11 0.06 0.70 800 183 12 D-011 72% 55 0.101.0 >1,000 550 10 D-012 69% 11 0.17 >588 65 D-013 71% 13 0.05 2.1 >2,000260 42 D-014 63% 3.6 0.06 0.56 >1,667 60 9.3 D-015 65% 69% 0.213.6 >480 >480 17 D-016 91% 81% 40 >2.5 >3 D-017 89% 108% 12 >8 >8 D-01888% 93% 4.2 >24 >24 D-019 67 105 7 10 15 D-020 69% 69% 1.9 >53 >53 D-021100 110 1.6 62 68 D-022 81% 110 0.8 40 >125 137.50 50 D-023 83% 91%26 >4 >3.9 D-024 100 76% 2.6 38 >38 D-025 73% 61% 0.11 1.5 >909 >909 14D-026 68% 54% 0.08 1.7 >1,250 >1,250 21 D-027 59% 58 0.6 >169 97 D-02867% 13 0.18 >556 69 D-029 49 3.0 0.06 882 54 D-030 50 5 0.07 758 70D-031 74 10 0.12 >833 83 D-034 19 11 0.15 131 74 D-035 9 3 0.05 199 65D-036 63% 31 0.4 >226 69 D-037 64% 80 0.8 >125 100 D-039 77% 66% 0.938 >111 >111 42 D-038 77% 63% 0.6 60 >167 >170 100 D-040 77% 64%1.7 >61 >61 D-041 67% 65% 4 >25 >25 D-042 70% 25 3 >32 8 D-043 83% 77%2.1 >47 >47 D-044 105 61 4.2 25 15 D-045 98% 74% 7.6 >13 >13 D-046 64%95 9 >11 11 D-047 30 9 0.09 0.5 333 100 5.6 D-048 70 14 0.16 449 90D-049 110% 30 1.0 >100 30 D-050 99% 41 1.6 >63 26 D-051 89% 57%3.3 >31 >31 D-052 0.7 69% 8 0.09 >13 D-121 69% 70% 0.48 >211 >211 D-999105 71% 47 60 2.2 2.1 1.3 LY294002 1.2 0.4 0.23 5.3 1.7 ¹⁾Compound D-121is 3-phenyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one

B. Biochemical Assay Using 200 μM ATP

In Part A, above, compounds of the invention were tested to establishtheir IC₅₀ for inhibition of the alpha, beta, delta, and gamma isoformsof PI3K using 20 μM ATP. A further screen was performed to establish theIC₅₀ for inhibition of the four PI3K isoforms at a final concentrationof 200 μM ATP, 10-fold greater, and substantially closer to the normalphysiological concentration of ATP in cells. This selectivity protocolis identical to that described above, except the 3× stock ATPconcentration was 600 μM. Data from this assay are summarized in Table3, below. The observed sensitivity to ATP concentration suggests thatthese PI3Kδ inhibitor compounds act as ATP competitors.

TABLE 3 Alpha/Delta Beta/Delta Gamma/Delta Compound Alpha IC₅₀ Beta IC₅₀Delta IC₅₀ Gamma IC₅₀ Ratio Ratio Ratio D-000 91 ± 1%   84 ± 2% 2 ± 1 35± 35 91 84 18 D-005 104%  82% 11      91% 20 16 17 D-006 104 ± 1%  44 ±5 0.92 ± 0.1  87 ± 33 226 48 95 D-007 92 ± 11%  72 ± 12 0.73 ± 0.2  88 ±4  252 99 121 D-009 70% 18    0.7 53 200 26 76 D-010 74 ± 18% 33 ± 40.23 ± 0.2  6 ± 3 658 144 27 D-011 88 ± 4%  105 ± 35 0.25 ± 0.2  61 ± 70700 420 244 D-012 70 ± 4%  108 ± 4  1.3 ± 0.4 50 ± 0  107 83 38 D-013117 ± 8%     73 ± 24% 0.51 ± 0.6  12 ± 1  461 289 24 D-014 100 ± 6%  13± 0 0.5 ± 0.4 5 ± 3 398 26 10 D-015 95 ± 22%  81 ± 3% 1.1 ± 0.5  83 ±37% 180 154 160 D-019 100%  100     30   33 7 3 1 D-022 88% 101%  4.2   60% 42 48 29 D-025 89 ± 11%  77 ± 6% 0.32 ± 0.3  7.8 ± 3  556 478 24D-026 83 ± 1%   77 ± 8% 0.38 ± 0.2  13 ± 10 443 411 34 D-027 74% 110    4   60 37 28 15 D-028 100%  81% 1.6 29 125 101 18 D-029 110 ± 12%  34 ±4 0.34 ± 0.08  13 ± 0.7 653 101 37 D-030 95 ± 11%  80 ± 14 0.53 ± 0.0531 ± 10 362 152 59 D-031 87 ± 10% 137 ± 23  0.2 ± 0.01 155 ± 60  903 707802 D-034 92 ± 11% 103 ± 4  1.2 ± 0.3 34 ± 1  153 85 28 D-035 95 ± 6   34 ± 6 0.49 ± 0.1  6.8 ± 1  193 69 14 D-036 99% 73% 4.1 72 48 36 18D-037 112%  58% 3.5 45 64 33 13 D-038 69% 74% 1.8 55 77 82 31 D-039 85%65% 2.6    57% 65 50 44 D-047 81% 30    0.2   4.5 810 150 23 D-048 90 ±57  95 ± 7 1.4 ± 0.9 123 ± 40  67 70 91 D-121 71% 62% 0.9    61% 158 138136 D-999 62% 71% 75   90 2 2 1 LY294002 23 ± 5    3.7 ± 2  2.1 ± 1.5 29± 13 11 2 13

EXAMPLE 12

Cell-Based Assay Data for Inhibitors of PI3δ Activity

Using the methods described in Examples 3-5, above, compounds of theinvention were tested for inhibitory activity and potency in assays ofstimulated B and T cell proliferation, neutrophil (PMN) migration, andneutrophil (PMN) elastase release. Data from these assays are set forthin Table 4, below. In Table 4, the values shown are effectiveconcentrations of the compound (EC₅₀; μM). Where no value is given, noassay was performed.

TABLE 4 Human PMN Human PMN Com- Mouse BCR Mouse TCE Elastase Migrationpound Stim (EC₅₀) Stim (EC₅₀) (EC₅₀) (EC₅₀) D-000 0.9 ± 0.4 5.5 ± 4  2.2 ± 2   1-5 D-003 3.9 5.7 D-005 0.7 ± 0.1 3.9 4.3 ± 1   D-006 0.2 ±0.1 5.3 0.3 ± 0.1 D-007 0.3 ± 0.1 4.2 0.4 D-008 1.0 D-009 0.3 ± 0.2 10.5D-010 0.2 ± 0.1 0.3 ± 0.3 D-011 0.3 ± 0.1 0.9 ± 0.7 D-012 0.3 ± 0.2 0.3D-013 1.4 D-014 0.2 ± 0.1 4.3 D-015 1.2 ± 0.2 1.8 1.3 ± 0.4 2.0 D-019 0.9 ± 0.01 0.9 D-021 1.8 3.5 D-022 1.8 2.3 D-024 2.9 D-025 0.3 ± 0.14.4 ± 0.6 0.3 ± 0.2 0.3 ± 0.3 D-026 0.3 ± 0.1 3.5 0.2 ± 0.2 0.3 ± 0.3D-027 >2     2 D-028 0.4 ± 0.2 1 D-029  0.1 ± 0.03 3.4 ± 2   0.5 ± 0.60.3 D-030 0.1 ± 0.1 6   0.4 ± 0.5 0.2 D-031 0.2 ± 0.1 0.7 ± 0.1 D-0340.6 ± 0.4 D-035 0.2 ± 0.1 2.9 ± 0.7 0.3 ± 0.1 D-036  0.9 ± 0.04 4.1 5.5± 5   0.2 D-037 1.2 ± 0.4 1.3 ± 0.4 2.0 D-038 1.4 ± 0.1 2.9 5 D-039 0.9± 0.1 5 D-043 1.4 2.6 D-045 9.0 D-047 0.3 ± 01. 0.5 ± 0.2 D-048 0.4 ±0.2 5   0.9 ± 0.2 D-049 2.0 6.3 5.0 D-121 1.4 D-999 3.1 ± 0.7 5.9 >201   LY294002 0.9 ± 0.5

EXAMPLE 13

Assay of Inhibitors of PI3Kδ Activity in Cancer Cells

The effect of compounds of the invention on cancer cell proliferationwas evaluated by testing one of the compounds against a panel of ChronicMyeloid Leukemia (CML) cell lines, including KU812, RWLeu4, K562, andMEG-01.

The inhibitory activity of the compound (D-000, dissolved in DMSO) wasdetermined as follows. The tested compound was added in a series ofconcentrations (0.001 μM to 20 μM) to 96-well microtiter plates withcells (1000 to 5000 cells/well). Plates were incubated for five days at37° C. during which the control cultures without test compound were ableto undergo at least two cell-division cycles. Cell growth was measuredby incorporation of [³H]-thymidine for eighteen hours added at daysthree, four, and five. Cells were transferred to a filter, washed andthe radioactivity counted using a Matrix 96 beta counter (Packard). Thepercentage of cell growth was measured as follows:

${\%\mspace{14mu}{Cell}\mspace{14mu}{growth}} = {\frac{\begin{pmatrix}{{a{verage}}\mspace{14mu}{counts}\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{incubated}\mspace{14mu}{with}\mspace{14mu} a} \\{{given}\mspace{14mu}{inhibitor}\mspace{14mu}{concentration}}\end{pmatrix} \times 100}{( {{average}\mspace{14mu}{counts}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{cells}\mspace{14mu}{without}\mspace{14mu}{inhibitor}} )}.}$The EC₅₀ value in these experiments was determined by the concentrationof the test compound that resulted in a radioactivity count 50% lowerthan that obtained using the control without inhibitor. The D-000compound exhibited inhibitory activity with an EC₅₀ of approximately 2μM for the KU812 and RWLeu4 lines. The compound was not found to exhibitan effect in the K562 and MEG-01 lines.

PI3Kδ inhibitors of the invention appear to inhibit CML cell growth andtherefore could be useful in the treatment of benign or malignanttumors. PI3Kδ expression has been demonstrated so far mostly in cells ofhematopoietic origin. However, it could be present in a broader varietyof proliferating cells. Therefore, the compounds of the invention couldbe used to induce tumor regression and to prevent the formation of tumormetastasis in both leukemia and solid tumor or in proliferation ofnontumoral origin. In addition, the compounds could be used both aloneand in combination with other pharmacologically active compounds or incombination with radiation as a sensitizing agent.

EXAMPLE 14

Measurement of Elastase Exocytosis in Mouse Air Pouch Lavage

The effect of D-030 on leukocyte influx and neutrophil elastaseexocytosis in animal models was tested. The six-day air pouch model isan in vivo inflammation model that histologically resembles a jointsynovium. A lining of organized mononuclear cells and fibroblastsdevelops that closely resembles a synovial cavity. The model representsan “acute” model of a chronic disease (e.g., rheumatoid arthritis). Thismodel allows for the in vivo evaluation of agents to block cellularinflux into the air pouch under the influence of an inflammatorystimulus.

The test was performed as follows: on day zero, groups of rats wereshaved and 10 ml of air was injected subcutaneously on the back of each,forming a pouch. On day three, 10 ml of air was reinjected. Six hoursprior to TNF challenge on day six, one group of rats (n=6) receivedD-030 (100 mg/kg in PEG 400 vehicle) orally, and another group (n=12)received vehicle alone orally. Six hours following dosing, the airpouches of both groups received 2.5 ng of TNF. Twelve hours followingdosing, the pouches were washed with saline, and the resulting lavagefluid was analyzed for leukocyte counts and neutrophil elastaseactivity. In addition, blood was drawn to determine the levels of D-030in circulation. The results were as follows: rats that received D-030for twelve hours had an average of 8.7 μM of compound in circulation andhad an 82% reduction in total leukocytes in the lavage fluid compared tovehicle controls. Reductions in specific leukocyte counts were asfollows: neutrophils (90%), eosinophils (66%), and lymphocytes (70%).Quantitation of neutrophil elastase showed that D-030-treated rats hadelastase levels that were somewhat reduced (15%) versus vehiclecontrols.

In another test, an area of the mouse back was shaved using clippers,and an air pouch was created by injecting 3 ml air subcutaneously. Onday three, the air injection was repeated. On day six, the animals weredosed with either D-030 (32 mg/kg in LABRAFIL®) or LABRAFIL® only onehour before and two hours after challenge with TNF-α (0.5 ng in 1 mlPBS), or PBS only. PBS is phosphate buffered saline. Four hours afterTNF challenge, the animals were anesthetized and the pouches werelavaged with 2 mL of 0.9% saline with 2 mM EDTA. The lavages werecentrifuged at 14,000 rpm in a microcentrifuge. Fifty microliters of thesupernatant was used to measure elastase exocytosis according to theprocedure described above.

As shown in FIG. 9, TNF challenge induced a high level of elastaseexocytosis compared to PBS challenged animals. However, when the TNFchallenged animals were treated with D-030, a significant decrease inthe elastase activity in the air pouch lavages was observed.

All publications and patent documents cited in this specification areincorporated herein by reference for all that they disclose.

While the present invention has been described with specific referenceto certain preferred embodiments for purposes of clarity andunderstanding, it will be apparent to the skilled artisan that furtherchanges and modifications can be practiced within the scope of theinvention as it is defined in the claims set forth below. Accordingly,no limitations should be placed on the invention other than thosespecifically recited in the claims.

What is claimed is:
 1. A compound of formula (I):

wherein A is a triazolyl ring optionally substituted with up to threesubstituents selected from the group consisting of N(R^(a))₂, halo,C₁₋₃alkyl, S(C₁₋₃alkyl), OR^(a), and

X is selected from the group consisting of C(R^(b))₂, CH₂CHR^(b), andCH═C(R^(b)); Y is selected from the group consisting of a single bond,S, SO, SO₂, NH, O, C(═O), OC(═O), C(═O)O, and NHC(═O)CH₂S; R¹ and R²,independently, are selected from the group consisting of hydrogen andC₁₋₆alkyl, aryl, heteroaryl, halo, NHC(═O)C₁₋₃alkyleneN(R^(a))₂, NO₂,OR^(a), CF₃, OCF₃, N(R^(a))₂, CN, OC(═O)R^(a), C(═O)R^(a), C(═O)OR^(a),arylOR^(b), Het, NR^(a)C(═O)C₁₋₃alkyleneC(═O)OR^(a),arylOC₁₋₃alkyleneN(R^(a))₂, arylOC(═O)R^(a), C₁₋₄alkyleneC(═O)OR^(a),OC₁₋₄alkyleneC(═O)OR^(a), C₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a),C(═O)NR^(a)SO₂R^(a), C₁₋₄alkyleneN(R^(a))₂, C₂₋₆alkenyleneN(R^(a))₂,C(═O)NR^(a)C₁₋₄alkyleneOR^(a), C(═O)NR^(a)C₁₋₄alkyleneHet,OC₂₋₄alkyleneN(R^(a))₂, OC₁₋₄alkyleneCH(OR^(b))CH₂N(R^(a))₂,OC₁₋₄alkyleneHet, OC₂₋₄alkyleneOR^(a), OC₂₋₄alkyleneNR^(a)C(═O)OR^(a),NR^(a)C₁₋₄alkyleneN(R^(a))₂, NR^(a)C(═O)R^(a), NR^(a)C(═O)N(R^(a))₂,N(SO₂C₁₋₄alkyl)₂, NR^(a)(SO₂C₁₋₄ alkyl), SO₂N(R^(a))₂, OSO₂CF₃,C₁₋₃alkylenearyl, C₁₋₄alkyleneHet, C₁₋₆alkyleneOR^(b),C₁₋₃alkyleneN(R^(a))₂, C(═O)N(R^(a))₂, NHC(═O)C₁-C₃alkylenearyl,C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl, arylOC₁₋₃alkyleneN(R^(a))₂,arylOC(═O)R^(b), NHC(═O)C₁₋₃alkyleneC₃₋₈heterocycloalkyl,NHC(═O)C₁₋₃alkyleneHet, OC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(b),C(═O)C₁₋₄alkyleneHet, and NHC(═O)haloC₁₋₆alkyl, each of which isoptionally substituted with a substituent selected from the groupconsisting of OCH₃, Cl, Br, F, CH₃, CF₃, NO₂, OH, N(CH₃)₂,

and O(CH₂)₂OCH₂C₆H₅; or R¹ and R² are taken together to form a 3- or4-membered alkylene or alkenylene chain component of a 5- or 6-memberedfused ring, optionally containing at least one heteroatom; R³ isselected from the group consisting of hydrogen, C₁₋₄alkylenearylsubstituted with one or more substituents selected from the groupconsisting of SO₂N(R^(a))₂, N(R^(a))₂, C(═O)OR^(a), NR^(a)SO₂CF₃, CN,NO₂, C(═O)R^(a), OR^(a), C₁₋₄alkyleneN(R^(a))₂, andOC₁₋₄alkyleneN(R^(a))₂, and C₁₋₆alkyl, C₃₋₈cycloalkyl,C₃₋₈heterocycloalkyl, C₁₋₄alkylenecycloalkyl, C₂₋₆alkenyl,C₁₋₃alkylenearyl, arylC₁₋₃alkyl, C(═O)R^(a), aryl, heteroaryl,C(═O)OR^(a), C(═O)N(R^(a))₂, C(═S)N(R^(a))₂, SO₂R^(a), SO₂N(R^(a))₂,S(═O)R^(a), S(═O)N(R^(a))₂, C(═O)NR^(a)C₁₋₄alkyleneOR^(a),C(═O)NR^(a)C₁₋₄alkyleneHet, C(═O)C₁₋₄alkylenearyl,C(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkyeneheteroaryl, C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)C₁₋₄alkylene-aryl, C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)C₁₋₄alkylene-aryl,C₁₋₄alkyleneC(═O)C₁₋₄alkyleneheteroaryl, C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneC(═O)N(R^(a))₂, C₁₋₄alkyleneOR^(a),C₁₋₄alkyleneNR^(a)C(═O)R^(a), C₁₋₄alkyleneOC₁₋₄alkyleneOR^(a),C₁₋₄alkyleneN(R^(a))₂, C₁₋₄alkyleneC(═O)OR^(a), andC₁₋₄alkyleneOC₁₋₄alkyleneC(═O)OR^(a), each of which is optionallysubstituted with a substituent selected from the group consisting ofhalo, OR^(a), C₁₋₆alkyl, aryl, heteroaryl, NO₂, N(R^(a))₂, NR^(a)SO₂CF₃,NR^(a)C(═O)R^(a), C(═O)OR^(a), SO₂N(R^(a))₂, CN, C(═O)R^(a),C₁₋₄alkyleneN(R^(a))₂, OC₁₋₄alkyleneC≡CR^(a),OC₁₋₄alkyleneC(═O)N(R^(a))₂, OC₁₋₄alkylenearyl, OC₁₋₄alkyleneheteroaryl,OC₁₋₄alkyleneHet, OC₁₋₄alkyleneN(R^(a))₂, andN(R^(a))C₁₋₄alkyleneN(R^(a))₂; each R^(a) is independently selected fromthe group consisting of hydrogen, C₁₋₆alkyl, C₃₋₈cycloalkyl,C₃₋₈heterocycloalkyl, C₁₋₃alkyleneN(R^(c))₂, aryl, arylC₁₋₃alkyl,C₁₋₃alkylenearyl, heteroaryl, heteroarylC₁₋₃alkyl, andC₁₋₃alkyleneheteroaryl; or two R^(a) groups are taken together to form a5- or 6-membered ring, optionally containing at least one heteroatom;each R^(b) is independently selected from the group consisting ofhydrogen, C₁₋₆alkyl, heteroC₁₋₃alkyl, C₁₋₃alkyleneheteroC₁₋₃alkyl,arylheteroC₁₋₃alkyl, aryl, heteroaryl, arylC₁₋₃alkyl,heteroarylC₁₋₃alkyl, C₁₋₃alkylenearyl, and C₁₋₃alkyleneheteroaryl; eachR^(c) is independently selected from the group consisting of hydrogen,C₁₋₆alkyl, C₃₋₈cycloalkyl, aryl, and heteroaryl; Het is a 5- or6-membered saturated or unsaturated heterocyclic ring, containing atleast one heteroatom, and optionally substituted with C₁₋₄alkyl orC(═O)OR^(a); wherein each heteroatom is independently selected from thegroup consisting of oxygen, nitrogen, and sulfur, or pharmaceuticallyacceptable salts or solvates thereof.
 2. The compound of claim 1 whereinX is selected from the group consisting of CH₂, CH₂CH₂, CH═CH, CH(CH₃),CH(CH₂CH₃), CH₂CH(CH₃), and C(CH₃)₂.
 3. The compound of claim 1 whereinX is selected from the group consisting of CH₂, CH(CH₃), and CH(CH₂CH₃).4. The compound of claim 1 wherein Y is selected from the groupconsisting of a single bond, O, S, and NH.
 5. The compound of claim 1wherein Y is a single bond, O or NH.
 6. The compound of claim 3 whereinY is a single bond, O or NH.
 7. The compound of claim 1 wherein A issubstituted with one to three substituents selected from the groupconsisting of N(R^(a))₂, halo, C₁₋₃alkyl, S(C₁₋₃alkyl), and OR^(a). 8.The compound of claim 1 wherein A is optionally substituted with one tothree substituents selected from the group consisting of NH₂, NH(CH₃),N(CH₃)₂, NHCH₂C₆H₅, NH(C₂H₅), Cl, F, CH₃, SCH₃, and OH.
 9. The compoundaccording to claim 1 wherein A is optionally substituted with NH₂. 10.The compound of claim 1 wherein R¹ and R², independently, are selectedfrom the group consisting of hydrogen, OR^(a), halo, C₁₋₆alkyl, CF₃,NO₂, N(R^(a))₂, and NR^(a)C₁₋₃alkyleneN(R^(a))₂, or R¹ and R² are takentogether to form a fused five- or six-membered ring.
 11. The compound ofclaim 1 wherein R¹ and R², independently, are selected from the groupconsisting of H, OCH₃, Cl, Br, F, CH₃, CF₃, NO₂, OH, N(CH₃)₂,O(CH₂)₂OCH₂C₆H₅, and


12. The compound according to claim 1 wherein R¹ and R², independently,are selected from the group consisting of H, Cl, or CH₃.
 13. Thecompound of claim 1 wherein R³ is selected from the group consisting ofC₁₋₆alkyl, aryl, heteroaryl, C₃₋₈cycloalkyl, C₃₋₈heterocycloalkyl,C(═O)OR^(a), C₁₋₄alkyleneHet, C₁₋₄alkylenecycloalkyl, C₁₋₄alkyleneC(═O)C₁₋₄alkylenearyl, C₁₋₄alkyleneC(═O)OR^(a), C₁₋₄alkyleneC(═O)N(R^(a))₂,C₁₋₄alkyleneC(═O)Het, C₁₋₄alkyleneN(R^(a))₂, andC₁₋₄alkyleneNR^(a)C(═O)R^(a), each of which is optionally substituted.14. The compound of claim 1 wherein R³ is selected from the groupconsisting of

each of which optionally substituted.
 15. The compound of claim 1wherein R³ is optionally substituted


16. The compound of claim 1 wherein R³ is substituted with a substituentselected from the group consisting of halo, OR^(a), C₁₋₆alkyl, aryl,heteroaryl, NO₂, N(R^(a))₂, NR^(a)SO₂CF₃, NR^(a)C(═O)R^(a), C(═O)OR^(a),SO₂N(R^(a))₂, CN, C(═O)R^(a), C₁₋₄alkyleneN(R^(a))₂,OC₁₋₄alkyleneC≡CR^(a), OC₁₋₄alkyleneC(═O)N(R^(a))₂, OC₁₋₄alkylenearyl,OC₁₋₄alkyleneheteroaryl, OC₁₋₄alkyleneHet, OC₁₋₄alkyleneN(R^(a))₂, andN(R^(a))C₁₋₄alkyleneN(R^(a))₂.
 17. The compound of claim 1 wherein R³ issubstituted with a substituent selected from the group consisting of Cl,F, CH₃, CH(CH₃)₂, OH, OCH₃, OCH₂C₆H₅, O(CH₂)₃N(CH₃)₂, OCH₂C≡CH,OCH₂C(═O)NH₂, C₆H₅, NO₂, NH₂, NHC(═O)CH₃, CO₂H, N(CH₃)CH₂CH₂N(CH₃)₂, and


18. A compound of formula (I):

wherein A is a triazolyl ring optionally substituted with up to threesubstituents selected from N(R^(a))₂, halo, C₁₋₃alkyl, S(C₁₋₃alkyl),OR^(a), and

X is selected from CH₂, CH₂CH₂, CH═CH, CH(CH₃), CH(CH₂CH₃), CH₂CH(CH₃),and C(CH₃)₂; Y is selected from the group consisting of a single bond,O, S and NH; R¹ and R², independently, are selected from the groupconsisting of hydrogen, OR^(a), CF₃, NO₂, N(R^(a))₂, halo,NR^(a)—C₁₋₃alkylene-N(R^(a))₂, and OC₁₋₃alkylene-OR^(a); and R³ isselected from the group consisting of

each of which is optionally substituted with up to three substituentsselected from the group consisting of Cl, F, CH₃, CH(CH₃)₂, OH, OCH₃,OCH₂C₆H₅, O(CH₂)₃N(CH₃)₂, OCH₂C≡CH, OCH₂C(═O)NH₂, C₆H₅, NO₂, NH₂,NHC(═O)CH₃, CO₂H, N(CH₃)CH₂CH₂N(CH₃)₂, and

each R^(a) is independently selected from the group consisting ofhydrogen and C₁₋₆alkyl; or two R^(a) groups are taken together to form a5- or 6-membered ring, optionally containing at least one heteroatomselected from the group consisting of oxygen, nitrogen, and sulfur; or apharmaceutically acceptable salt thereof.
 19. The compound of claim 18wherein X is selected from the group consisting of CH₂, CH(CH₃), andCH(CH₂CH₃).
 20. The compound of claim 19 wherein Y is a single bond. 21.The compound of claim 19, wherein Y is NH.
 22. The compound of claim 19wherein Y is O.
 23. The compound according to claim 18 wherein A issubstituted with one or two substituents.
 24. The compound of claim 18wherein A is optionally substituted with one to three substituentsselected from the group consisting of NH₂, NH(CH₃), N(CH₃)₂, NHCH₂C₆H₅,NH(C₂H₅), Cl, F, CH₃, SCH₃, and OH.
 25. The compound according to claim18 wherein A is substituted with NH₂.
 26. The compound according toclaim 18 wherein R¹ and R², independently, are selected from the groupconsisting of H, Cl, and CH₃.
 27. The compound of claim 18 wherein R³ isoptionally substituted


28. The compound of claim 18 wherein R³ is substituted with asubstituent selected from the group consisting of Cl, F, CH₃, CH(CH₃)₂,OH, OCH₃, OCH₂C₆H₅, O(CH₂)₃N(CH₃)₂, OCH₂C≡CH, OCH₂C(═O)NH₂, C₆H₅, NO₂,NH₂, NHC(═O)CH₃, CO₂H, N(CH₃)CH₂CH₂N(CH₃)₂, and


29. The compound of claim 28, wherein A is optionally substituted with1-2substituents selected from NH₂, NH(CH₃), N(CH₃)₂, NHCH₂C₆H₅,NH(C₂H₅), Cl, F, CH₃, SCH₃, and OH.
 30. A pharmaceutical compositioncomprising: a) an effective amount of a compound of formula (I):

wherein A is a triazolyl ring optionally substituted with up to threesubstituents selected from N(R^(a))₂, halo, C₁₋₃alkyl, S(C₁₋₃alkyl),OR^(a), and

X is selected from CH₂, CH₂CH₂, CH═CH, CH(CH₃), CH(CH₂CH₃), CH₂CH(CH₃),and C(CH₃)₂; Y is selected from the group consisting of a single bond,O, S and NH; R¹ and R², independently, are selected from the groupconsisting of hydrogen, OR^(a), CF₃, NO₂, N(R^(a))₂, halo,NR^(a)—C₁₋₃alkylene-N(R^(a))₂, and OC₁₋₃alkylene-OR^(a); R³ is selectedfrom the group consisting of

each of which is optionally substituted with up to three substituentsselected from the group consisting of Cl, F, CH₃, CH(CH₃)₂, OH, OCH₃,OCH₂C₆H₅, O(CH₂)₃N(CH₃)₂, OCH₂C≡CH, OCH₂C(═O)NH₂, C₆H₅, NO₂, NH₂,NHC(═O)CH₃, CO₂H, N(CH₃)CH₂CH₂N(CH₃)₂, and

each R^(a) is independently selected from the group consisting ofhydrogen and C₁₋₆alkyl; or two R^(a) groups are taken together to form a5- or 6-membered ring, optionally containing at least one heteroatomselected from the group consisting of oxygen, nitrogen, and sulfur; andor a pharmaceutically acceptable salt thereof.